Novel compositions with polymerase activity

ABSTRACT

The invention provides novel compositions with polymerase activity and methods of using the compositions.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/627,582 filed Jun. 25, 2003, which claims the benefit of U.S.Provisional Application No. 60/398,687 filed Jul. 25, 2002, and U.S.Provisional Application 60/483,287 filed Jun. 27, 2003, each of whichapplications is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention provides novel compositions with polymeraseactivity and methods of using those compositions

BACKGROUND OF THE INVENTION

Polymerases catalyze the formation of biological polymers. Polymerasesare useful for the synthesis of DNA from deoxyribonucleosidetriphosphates in the presence of a nucleic acid template and a nucleicacid primer; the synthesis of RNA from ribonucleotides and a DNA or RNAtemplate; DNA replication and repair; and in vitro DNA or RNAamplification.

The 3′ to 5′ exonuclease activity, commonly referred to as“proofreading” activity, is an important characteristic of some DNApolymerases and is present in Pyrococcus species family B polymerasessuch as Pyrococcus furiosus PolI (referred to herein as “Pfu” anddescribed in U.S. Pat. No. 5,948,663; commercially available fromStratagene, San Diego, Calif.) and Pyrococcus strain GB-D PolI (referredto herein as “Deep Vent®” and described U.S. Pat. No. 5,834,285;commercially available from New England Biolabs, Beverly Mass.). Theessential function of the 3′ to 5′ exonuclease is to recognize andcleave a non-base-paired terminus. Enzymes with high exonucleaseactivity, however, are not commonly used in reactions relying onpolymerase activity because they have poor processivity. For example, ifused in PCR, it is often in combination with Thermus aquaticus DNA PolI,(Taq), an enzyme with higher processivity but no 3′ to 5′ exonucleaseactivity, in order to improve the fidelity of the PCR reaction. Improvedprocessivity in polymerases with high 3′ to 5′ exonuclease activitywould greatly increase the reliability of reactions relying on the useof polymerases and would eliminate, in some cases, the need for Taqpolymerase. Accordingly, a need exists for creating improved polymeraseswith 3′ to 5′ exonuclease activity.

This invention addresses this and other needs by providing novelcompositions with polymerase activity.

BRIEF SUMMARY OF THE INVENTION

The invention provides hybrid polymerase polypeptides having residuesfrom multiple parent polymerases. The invention also provides nucleicacids encoding such proteins. Thus, in one aspect, the inventionprovides a hybrid polymerase having polymerase activity, wherein thepolymerase comprises SEQ ID NO:23 and is at least 80% identical over 700contiguous amino acids of the Pyrococcus furiosus (Pfu) sequence setforth in SEQ ID NO: 24 or at least 80% identical over 700 contiguousamino acids of the Deep Vent® sequence set forth in SEQ ID NO:25, withthe proviso that (a) when the polymerase is at least 85% identical toSEQ ID NO:24, the sequence comprises at least one hybrid position thatis mutated from the native Pfu residue to the residue that occurs at thecorresponding position of SEQ ID NO:25, wherein the hybrid position isone of the residues designated as “X” in SEQ ID NO:26; or (b) when thepolymerase is at least 85% identical to SEQ ID NO:25, the sequencecomprises at least one hybrid position that is mutated from the nativeDeep Vent® residue to the residue that occurs at the correspondingposition of SEQ ID NO:24, wherein the hybrid position is one of theresidues designated as “X” in SEQ ID NO:26. In some embodiments, thepolymerase is at least 90% identical over 700 contiguous amino acids ofthe Pfu sequence set forth in SEQ ID NO:24 or at least 90% identicalover 700 contiguous amino acids of the Deep Vent® sequence set forth inSEQ ID NO:25.

In some embodiments, the hybrid polymerase comprises at least ten hybridpositions, typically at least twenty hybrid positions, or at leastthirty hybrid positions, or at least forty hybrid positions, or at leastfifty or more hybrid positions, that are mutated from the native resideof SEQ ID NO:24 or SEQ ID NO:25 to the corresponding residue of SEQ IDNO:25 or SEQ ID NO:24, respectively.

In other embodiments, the hybrid polymerase comprises an amino acidsequence of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18; orthe polymerase region of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:14, or SEQ ID NO:20

The invention also includes embodiments in which the hybrid polymerasefurther comprises a DNA binding domain, often Sso7d, Sac7d, and Sac7e.Often, the DNA binding domain is conjugated to the polymerase. In someembodiments, the polymerase DNA binding domain conjugate comprises anamino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:14, or SEQ ID NO:20.

The invention also provides isolated nucleic acids encoding the hybridpolymerases, and conjugates comprising the hybrid polymerase linked to aDNA binding domain; and expression vectors and host cells comprising thenucleic acids.

In another aspect, the invention provides an isolated nucleic acidencoding a polypeptide comprising an amino acid sequence at least 94%identical to SEQ ID NO:2, wherein the polypeptide exhibits polymeraseactivity. In typical embodiments, the polypeptide comprises SEQ ID NO:2.In some embodiments, the isolated nucleic acid comprises SEQ ID NO:1.

The invention also provides embodiments, wherein the polypeptide encodedby the nucleic acid further comprises a DNA binding domain, which isoften selected from the group consisting of Sso7d, Sac7d, and Sac7e. Thenucleic acid can encode a polypeptide comprising SEQ ID NO:4. In oneembodiment, the nucleic acid comprises SEQ ID NO:3.

In other aspects, the invention provides expression vectors and hostcells comprising the nucleic acids.

In another aspect, the invention provides an isolated polypeptidecomprising an amino acid sequence at least 94% identical to SEQ ID NO:2,wherein the polypeptide has polymerase activity. In one embodiment, thepolypeptide comprises SEQ ID NO:2.

In some embodiments, the polypeptide further comprises a DNA-bindingdomain, e.g., Sso7d, Sac7d, or Sac7e. The DNA-binding domain can befused to the carboxy-terminus of the polypeptide. In one embodiment, thepolypeptide comprises SEQ ID NO:4.

In another aspect, the invention provides an isolated nucleic acidencoding a polypeptide comprising an amino acid sequence at least 94%identical to SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18; or thepolymerase region of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:14, or SEQ ID NO:20, wherein the polypeptide exhibits polymeraseactivity. In typical embodiments, the polypeptide comprises SEQ IDNO:12, SEQ ID NO:16, or SEQ ID NO:18; or the polymerase region of SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:20. In someembodiments, the isolated nucleic acid comprises SEQ ID NO:11, SEQ IDNO:15, or SEQ ID NO:17; or the polymerase region of SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:19.

The invention also provides embodiments, wherein the polypeptide encodedby the nucleic acid further comprises a DNA binding domain, which isoften selected from the group consisting of Sso7d, Sac7d, and Sac7e. Thenucleic acid can encode a polypeptide comprising SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:20. In one embodiment,the nucleic acid comprises SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:13, or SEQ ID NO:1.

In other aspects, the invention provides expression vectors and hostcells comprising the nucleic acids.

In another aspect, the invention provides an isolated polypeptidecomprising an amino acid sequence at least 94% identical to SEQ IDNO:12, SEQ ID NO:16, or SEQ ID NO:18; or the polymerase region of SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:20, whereinthe polypeptide has polymerase activity. In one embodiment, thepolypeptide comprises SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18; orthe polymerase region of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:14, or SEQ ID NO:20.

In some embodiments, the further comprising a DNA binding domain, e.g.,Sso7d, Sac7d, or Sac7e. The DNA binding domain can be fused to thecarboxy-terminus of the polypeptide. In one embodiment, the polypeptidecomprises SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, or SEQID NO:20.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the parent Pfu (SEQ ID NO:24) and DeepVent® (SEQ ID NO:25) polymerase sequences. The hybrid protein designpolymerase sequence 9 SEQ ID NO:27) shows the positions that vary,between the two parent sequences, which are designated by an X.“Corresponding residues” in the sequences are those residues that occurin the same position as shown in the alignment.

FIG. 2 shows assembly PCR of sequences encoding hybrid polymerases. Inthis example, 100 base pair degenerate oligonucleotides are subjected torounds of annealing and primer extension until fragments ofapproximately 500 base pairs are obtained. These fragment libraries aresufficiently large in size to be easily manipulated and assembled intofull length clones or libraries of full length clones by conventionalmolecular cloning techniques.

FIG. 3 shows a comparison of the polymerase to 3′ exonuclease ratios forseveral commercially available enzymes, including the parental proteins,and isolates from the hybrid library.

FIG. 4 shows the results of a comparison of hybrid and parentpolymerases. The enzymes were tested for the ability to amplifybacteriophage lambda DNA amplicons of a range of sizes, given a 30 secor 1 min extension time. The sizes of the amplicons, in kilobases, arelisted across the bottom of the lanes. Twenty units of enzyme per mlwere used unless otherwise specified.

FIG. 5 shows an alignment of the parental Pfu (SEQ ID NO:24) and DeepVent® (SEQ ID NO:25) sequences, and various hybrid polymerase sequences(Hybrid_design, HyS1, Hyb2, Hyb3, HyS4, PhS1, PhS2, PhS3, PhS4, PhS5,PhS6, and PhS7; SEQ ID NOS:27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,and 10, respectively).

FIG. 6 shows a sequence element that is common to the parental andhybrid sequences Pfu (SEQ ID NO:38); Deep Vent (SEQ ID NO:39); Hybirdfinal protein (SEQ ID NO:40); HyS1, Hyb2, Hyb3, and HyS4 (SEQ ID NO:41);PhS1 (SEQ ID NO:42); PhS2 (SEQ ID NO:43); PhS4 (SEQ ID NO:44); PhS5 (SEQID NO:45); and PhS7 (SEQ ID NO:46).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “hybrid polymerase” is used herein to describe a polymerasethat comprises amino acid residues from multiple parent sequences.

The term “hybrid position” refers to a position that differs betweenparent polymerase sequences, or subsequences.

A “wild type polymerase” refers to a naturally occurring polymerase. A“wild type polymerase amino acid sequence” refers to the naturallyoccurring amino acid sequence.

A “native” polymerase sequence refers to a parent polymerase sequence,typically a “wildtype” sequence.

A “parent polymerase sequence” indicates a starting or reference aminoacid or nucleic acid sequence prior to a manipulation of the invention.The term is used interchangeably with “starting sequence”. Parentsequences may be wild-type proteins, proteins containing mutations, orother engineered proteins. Parent sequences can also be full-lengthproteins, protein subunits, protein domains, amino acid motifs, proteinactive sites, or any polymerase sequence or subset of polymerasesequences, whether continuous or interrupted by other polypeptidesequences.

The term “DNA binding domain” refers to a protein domain which bindswith significant affinity to DNA, for which there is no known nucleicacid which binds to the protein domain with more than 100-fold moreaffinity than another nucleic acid with the same nucleotide compositionbut a different nucleotide sequence.

The term “Sso7d” or “Sso7d DNA binding domain” or “Sso7d-like DNAbinding domain” or “Sso7d binding protein” refers to nucleic acid andpolypeptide polymorphic variants, alleles, mutants, and interspecieshomologs that: (1) have an amino acid sequence that has greater thanabout 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%,preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino acid sequence identity, preferably over a region of at least about15, 25, 35, 50, or more amino acids, to an Sso7d sequence of SEQ IDNO:22; (2) bind to antibodies, e.g., polyclonal antibodies, raisedagainst an immunogen comprising an amino acid sequence of SEQ ID NO:22and conservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to a Sso7d nucleic acidsequence of SEQ ID NO:21 and conservatively modified variants thereof;or (4) have a nucleic acid sequence that has greater than about 90%,preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotidesequence identity, preferably over a region of at least about 50, 100,150, or more nucleotides, to SEQ ID NO:21. The term includes bothfull-length Sso7d polypeptides and fragments of the polypeptides thathave sequence non-specific double-stranded binding activity. Sso7d-likeproteins include Sac7d and Sac7e.

“Domain” refers to a unit of a protein or protein complex, comprising apolypeptide subsequence, a complete polypeptide sequence, or a pluralityof polypeptide sequences where that unit has a defined function. Thefunction is understood to be broadly defined and can be ligand binding,catalytic activity or can have a stabilizing effect on the structure ofthe protein.

An “Sso7d polymerase conjugate” refers to a modified polymerasecomprising at least one Sso7D DNA binding domain joined to a polymerasedomain, or a catalytic subunit of the polymerase domain.

“Efficiency” in the context of a polymerase of this invention refers tothe ability of the enzyme to perform its catalytic function underspecific reaction conditions. Typically, “efficiency” as defined hereinis indicated by the amount of product generated under given reactionconditions.

“Enhances” in the context of an enzyme refers to improving the activityof the enzyme, i.e., increasing the amount of product per unit enzymeper unit time.

“Fused” refers to linkage by covalent bonding.

“Heterologous”, when used with reference to portions of a protein,indicates that the protein comprises two or more domains that are notfound in the same relationship to each other in nature. Such a protein,e.g., a fusion protein, contains two or more domains from unrelatedproteins arranged to make a new functional protein.

“Join” refers to any method known in the art for functionally connectingprotein domains, including without limitation recombinant fusion with orwithout intervening domains, intein-mediated fusion, non-covalentassociation, and covalent bonding, including disulfide bonding; hydrogenbonding; electrostatic bonding; and conformational bonding, e.g.,antibody-antigen, and biotin-avidin associations.

“Polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides. The term encompasses both the full lengthpolypeptide and a domain that has polymerase activity.

“Processivity” refers to the ability of a polymerase to remain bound tothe template or substrate and perform polynucleotide synthesis.Processivity is measured by the number of catalytic events that takeplace per binding event.

“Thermally stable polymerase” as used herein refers to any enzyme thatcatalyzes polynucleotide synthesis by addition of nucleotide units to anucleotide chain using DNA or RNA as a template and has an optimalactivity at a temperature above 45° C.

“Thermus polymerase” refers to a family A DNA polymerase isolated fromany Thermus species, including without limitation Thermus aquaticus,Thermus brockianus, and Thermus thermophilus; any recombinantpolymerases deriving from Thermus species, and any functionalderivatives thereof, whether derived by genetic modification or chemicalmodification or other methods known in the art.

The term “amplification reaction” refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid. Suchmethods include but are not limited to polymerase chain reaction (PCR),DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)), (LCR), QBeta RNA replicase, and RNA transcription-based (such asTAS and 3SR) amplification reactions as well as others known to those ofskill in the art.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a polynucleotide if all of thecomponents of the reaction are intact. Components of an amplificationreaction include, e.g., primers, a polynucleotide template, polymerase,nucleotides, and the like. The term “amplifying” typically refers to an“exponential” increase in target nucleic acid. However, “amplifying” asused herein can also refer to linear increases in the numbers of aselect target sequence of nucleic acid, such as is obtained with cyclesequencing.

The term “amplification reaction mixture” refers to an aqueous solutioncomprising the various reagents used to amplify a target nucleic acid.These include enzymes, aqueous buffers, salts, amplification primers,target nucleic acid, and nucleoside triphosphates. Depending upon thecontext, the mixture can be either a complete or incompleteamplification reaction mixture

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990. Exemplary PCR reaction conditions typically comprise either two orthree step cycles. Two step cycles have a denaturation step followed bya hybridization/elongation step. Three step cycles comprise adenaturation step followed by a hybridization step followed by aseparate elongation step.

“Long PCR” refers to the amplification of a DNA fragment of 5 kb or morein length. Long PCR is typically performed using specially-adaptedpolymerases or polymerase mixtures (see, e.g., U.S. Pat. Nos. 5,436,149and 5,512,462) that are distinct from the polymerases conventionallyused to amplify shorter products.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths and areoften less than 50 nucleotides in length, for example 12-30 nucleotides,in length. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art, see,e.g., Innis et al., supra.

A “temperature profile” refers to the temperature and lengths of time ofthe denaturation, annealing and/or extension steps of a PCR or cyclesequencing reaction. A temperature profile for a PCR or cycle sequencingreaction typically consists of 10 to 60 repetitions of similar oridentical shorter temperature profiles; each of these shorter profilesmay typically define a two step or three-step cycle. Selection of atemperature profile is based on various considerations known to those ofskill in the art, see, e.g., Innis et al., supra. In a long PCR reactionas described herein, the extension time required to obtain anamplification product of 5 kb or greater in length is reduced comparedto conventional polymerase mixtures.

PCR “sensitivity” refers to the ability to amplify a target nucleic acidthat is present in low copy number. “Low copy number” refers to 10⁵,often 10⁴, 10³, 10², 10¹ or fewer, copies of the target sequence in thenucleic acid sample to be amplified.

The term “polymerase primer/template binding specificity” as used hereinrefers to the ability of a polymerase to discriminate between correctlymatched primer/templates and mismatched primer templates. An “increasein polymerase primer/template binding specificity” in this contextrefers to an increased ability of a polymerase of the invention todiscriminate between matched primer/template in comparison to a wildtype polymerase protein.

A “template” refers to a double stranded polynucleotide sequence thatcomprises the polynucleotide to be amplified, flanked by primerhybridization sites. Thus, a “target template” comprises the targetpolynucleotide sequence flanked by hybridization sites for a 5′ primerand a 3′ primer.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-RUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

For example, substitutions may be made wherein an aliphatic amino acid(G, A, I, L, or V) is substituted with another member of the group.Similarly, an aliphatic polar-uncharged group such as C, S, T, M, N, orQ, may be substituted with another member of the group; and basicresidues, e.g., K, R, or H, may be substituted for one another. In someembodiments, an amino acid with an acidic side chain, E or D, may besubstituted with its uncharged counterpart, Q or N, respectively; orvice versa. Each of the following eight groups contains other exemplaryamino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

The term “encoding” refers to a polynucleotide sequence encoding one ormore amino acids. The term does not require a start or stop codon. Anamino acid sequence can be encoded in any one of six different readingframes provided by a polynucleotide sequence.

The term “promoter” refers to regions or sequence located upstreamand/or downstream from the start of transcription and which are involvedin recognition and binding of RNA polymerase and other proteins toinitiate transcription.

A “vector” refers to a polynucleotide, which when independent of thehost chromosome, is capable replication in a host organism. Preferredvectors include plasmids and typically have an origin of replication.Vectors can comprise, e.g., transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular nucleic acid.

“Recombinant” refers to a human manipulated polynucleotide or a copy orcomplement of a human manipulated polynucleotide. For instance, arecombinant expression cassette comprising a promoter operably linked toa second polynucleotide may include a promoter that is heterologous tothe second polynucleotide as the result of human manipulation (e.g., bymethods described in Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)or Current Protocols in Molecular Biology Volumes 1-3, John Wiley &Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising theexpression cassette. In another example, a recombinant expressioncassette may comprise polynucleotides combined in such a way that thepolynucleotides are extremely unlikely to be found in nature. Forinstance, human manipulated restriction sites or plasmid vectorsequences may flank or separate the promoter from the secondpolynucleotide. One of skill will recognize that polynucleotides can bemanipulated in many ways and are not limited to the examples above.

A “polymerase nucleic acid” or “polymerase polynucleotide” is apolynucleotide sequence or subsequence encoding a polymerase. Exemplarypolymerase nucleic acids of the invention are identical or substantiallyidentical to a polymerase sequence set forth in SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19; which encodes a polymerasepolypeptide identical or substantially identical to SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20.

A “polymerase polypeptide” of the present invention is a proteincomprising a polymerase domain. The polymerase polypeptide may alsocomprise additional domains including a DNA binding domain, e.g., Sso7D.DNA polymerases are well-known to those skilled in the art, e.g.,Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima.They include both DNA-dependent polymerases and RNA-dependentpolymerases such as reverse transcriptase. At least five families ofDNA-dependent DNA polymerases are known, although most fall intofamilies A, B and C. There is little or no sequence similarity among thevarious families. Most family A polymerases are single chain proteinsthat can contain multiple enzymatic functions including polymerase, 3′to 5′ exonuclease activity and 5′ to 3′ exonuclease activity. Family Bpolymerases typically have a single catalytic domain with polymerase and3′ to 5′ exonuclease activity, as well as accessory factors. Family Cpolymerases are typically multi-subunit proteins with polymerizing and3′ to 5′ exonuclease activity. In E. coli, three types of DNApolymerases have been found, DNA polymerases I (family A), II (familyB), and III (family C). In eukaryotic cells, three different family Bpolymerases, DNA polymerases α, δ, and ε, are implicated in nuclearreplication, and a family A polymerase, polymerase γ, is used formitochondrial DNA replication. Other types of DNA polymerases includephage polymerases. Similarly, RNA polymerases typically includeeukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerasesas well as phage and viral polymerases. RNA polymerases can beDNA-dependent and RNA-dependent.

Exemplary embodiments of polymerases of the present invention include apolymerase identical or substantially identical to SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20. A skilledpractitioner will understand that specific amino acid residues withinthe polymerases can be modified, e.g., conservatively modified, withoutsignificantly affecting the improved polymerase ability. On average,there are at least 6 amino acids per 100 that can be modified. Theyinclude, for example, replacing glycine at position 12 with alanine,methionine at position 1 with valine, isoleucine at position 2 withleucine, isoleucine at position 8 with valine, or threonine at position33 with serine. (Positions are indicated with reference to SEQ IDNO:26.)

The polymerases of the present invention may be identified by theirability to bind to antibodies, e.g., polyclonal antibodies, raisedagainst an immunogen comprising an amino acid sequence of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 and conservativelymodified variants thereof.

Polypeptide polymerases of the present invention have polymeraseactivity. Using the assays described herein, the activity of thepolypeptides of the present invention can be measured. Some polymerasepolypeptides of the invention exhibit improved polymerase activity ascompared to wild type polymerases in the assays described herein.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The terms “identical” or percent “identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence over acomparison window, as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Whenpercentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity. More preferred embodiments include at least: 75%, 80%, 85%,90%, 94%, 95%, 96%, 97%, 98%, or 99% compared to a reference sequenceusing the programs described herein; preferably BLAST using standardparameters, as described below. One of skill will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 94%. Morepreferred embodiments include at least 94%, 95%, 96%, 97%, 98% or 99%.Polypeptides which are “substantially similar” share sequences as notedabove except that residue positions which are not identical may differby conservative amino acid changes. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.The term “at least 94% identical” refers to a sequence that is at least94%, possibly 95%, 96%, 97%, 98%, 99% or 100% identical to a referencesequence.

One of skill in the art will recognize that two polypeptides can also be“substantially identical” if the two polypeptides are immunologicallysimilar. Thus, overall protein structure may be similar while theprimary structure of the two polypeptides display significant variation.Therefore a method to measure whether two polypeptides are substantiallyidentical involves measuring the binding of monoclonal or polyclonalantibodies to each polypeptide. Two polypeptides are substantiallyidentical if the antibodies specific for a first polypeptide bind to asecond polypeptide with an affinity of at least one third of theaffinity for the first polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Extension of the word hits in each direction are halted when:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST algorithm parametersW, T, and X determine the sensitivity and speed of the alignment. TheBLAST program uses as defaults a word length (W) of 11, the BLOSUM62scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4,and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, highly stringent conditions are selected to be about 5-10° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength pH. Low stringency conditions are generallyselected to be about 15-30° C. below the T_(m). The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Hybridization conditions are typically those in which thesalt concentration is less than about 1.0 M sodium ion, typically about0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g., 10to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. Forselective or specific hybridization, a positive signal is at least twotimes background, preferably 10 times background hybridization.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Exemplary “stringent hybridizationconditions” include a hybridization in a buffer of 40% formamide, 1 MNaCl, 1% SDS at 37° C., and at least one wash in 0.2×SSC at atemperature of at least about 50° C., usually about 55° C. to about 60°C., for 20 minutes, or equivalent conditions. A positive hybridizationis at least twice background. Those of ordinary skill will readilyrecognize that alternative hybridization and wash conditions can beutilized to provide conditions of similar stringency.

Introduction

The present invention provides novel polymerase polypeptide and nucleiccid sequences. In some embodiments, the polypeptides further comprise aDNA binding domain, e.g., an Archaeal small basic protein, such as anSso7d, Sac7d, or Sac7e DNA binding domain, which is fused to thepolypeptide. The DNA binding domain typically increases the bindingaffinity of the enzyme to nucleic acid and can enhance the processivityof the polymerases.

A polymerase of the invention includes polymerases identical orsubstantially identical to the polymerase sequences disclosed in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20.

Polymerases of the invention exhibit the same or altered polymeraseactivity compared to that of wild type polymerase, e.g., Pfu or DeepVent® polymerase, in accordance with the activity assays describedherein.

Generation of Nucleic Acids Encoding Polymerases

Polymerases of the present invention can be produced by methods known tothose of skill in the art. For example, the nucleic acid sequencesencoding a Phy1 or PhS1 polymerase of the invention are provided as SEQID NO:1 and SEQ ID NO:3 and the amino acid sequences of a Phy1 or PhS1polymerase are provided as SEQ ID NO:2 and SEQ ID NO:4. Polymerases, orsubsequences thereof, may be synthesized using recombinant DNAmethodology. Generally this involves creating a DNA sequence thatencodes the polypeptide, modified as desired, placing the DNA in anexpression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if required, renaturing the protein.

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers et al.,Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams etal., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or bysynthesizing the complementary strand using DNA polymerase with anappropriate primer sequence. Polymerases may also be ordered from avariety of commercial sources known to persons of skill.

Assembly PCR can be used, in a process that involves the assembly of aPCR product from a mixture of small DNA fragments. A large number ofdifferent PCR reactions can occur in parallel in the same reactionmixture, with the products of one reaction priming the products ofanother reaction. Alternatively, the skilled practitioner, usingassembly PCR, can completely synthesize the claimed nucleotidesequences.

B. Generation of a Polymerase Nucleic Acid by Modification of Wild Type

Wild type polymerase nucleic acids may be isolated from naturallyoccurring sources to be used as starting material to generate novelpolymerases. Generally, the nomenclature and the laboratory proceduresin recombinant DNA technology described below are those well known andcommonly employed in the art. Standard techniques for cloning, DNA andRNA isolation, amplification and purification are known. Generallyenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases are the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook & Russell, Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, JohnWiley & Sons, Inc. (1994-1998) (“Ausubel et al.”).

The isolation of polymerase nucleic acids may be accomplished by avariety of techniques. For instance, genes encoding Pfu or Deep Vent®can be constructed as described in U.S. Pat. Nos. 5,948,663 and5,834,285.

The polymerase nucleic acids of the present invention can be generatedfrom the wild type sequences. The wild type sequences are altered tocreate modified sequences. Wild type polymerases can be readily modifiedto create the polymerases claimed in the present application usingmethods that are well known in the art. Exemplary modification methodsare site-directed mutagenesis, point mismatch repair, oroligonucleotide-directed mutagenesis. Polymerase polynucleotides of theinvention, e.g., SEQ ID NO:1 or SEQ ID NO:3, can also be readily alteredusing these modification methods.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that manymodification techniques exist and are often not mutually exclusive.Indeed, the various methods can be used singly or in combination, inparallel or in series, to access polymerases of the present invention.

The result of any of the modification procedures described herein can bethe generation of one or more nucleic acids, which can be selected orscreened for nucleic acids that encode proteins with polymeraseactivity. Following modification of a polymerase, e.g., a wild typepolymerase, or hybrid polymerase such as SEQ ID NO:2 or SEQ ID NO:4, byone or more of the methods herein, or otherwise available to one ofskill, any nucleic acids that are produced can be selected for a desiredactivity or property, e.g. polymerase activity. This can includeidentifying any activity that can be detected by any of the assays knownin the art for determining polymerase activity.

Site directed mutagenesis is well known in the art and is described inthe following references, e.g., (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein (1986) “Inhibitionof restriction endonuclease Nci I cleavage by phosphorothioate groupsand its application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

An additional modification method well known in the art is pointmismatch repair, e.g., (Kramer et al. (1984) “Point Mismatch Repair”Cell 38:879-887), mutagenesis using repair-deficient host strains(Carter et al. (1985) “Improved oligonucleotide site-directedmutagenesis using M13 vectors” Nucl. Acids Res. 13: 4431-4443; andCarter (1987) “Improved oligonucleotide-directed mutagenesis using M13vectors” Methods in Enzymol. 154: 382-403), deletion mutagenesis(Eghtedarzadeh & Henikoff (1986) “Use of oligonucleotides to generatelarge deletions” Nucl. Acids Res. 14: 5115), restriction-selection andrestriction-selection and restriction-purification (Wells et al. (1986)“Importance of hydrogen-bond formation in stabilizing the transitionstate of subtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423),mutagenesis by total gene synthesis (Nambiar et al. (1984) “Totalsynthesis and cloning of a gene coding for the ribonuclease S protein”Science 223: 1299-1301; Sakamar and Khorana (1988) “Total synthesis andexpression of a gene for the a-subunit of bovine rod outer segmentguanine nucleotide-binding protein (transducing)” Nucl. Acids Res. 14:6361-6372; Wells et al. (1985) “Cassette mutagenesis: an efficientmethod for generation of multiple mutations at defined sites” Gene34:315-323; and Grundström et al. (1985) “Oligonucleotide-directedmutagenesis by microscale ‘shot-gun’ gene synthesis” Nucl. Acids Res.13: 3305-3316), double-strand break repair (Mandecki (1986); Arnold(1993) “Protein engineering for unusual environments” Current Opinion inBiotechnology 4:450-455. “Oligonucleotide-directed double-strand breakrepair in plasmids of Escherichia coli: a method for site-specificmutagenesis” Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additionaldetails on many of the above methods can be found in Methods inEnzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Oligonucleotide directed mutagenesis could be used to introducesite-specific mutations in a nucleic acid sequence of interest. Examplesof such techniques are found in the references above and, e.g., inReidhaar-Olson et al. (1988) Science 241:53-57. Similarly, cassettemutagenesis can be used in a process that replaces a small region of adouble stranded DNA molecule with a synthetic oligonucleotide cassettethat differs from the native sequence. The oligonucleotide can contain,e.g., completely and/or partially randomized native sequence(s).

Modification of Polymerase Nucleic Acids for Common Codon Usage in anOrganism

The polynucleotide sequence encoding a particular polymerase can bealtered to coincide with the codon usage of a particular host. Forexample, the codon usage of E. coli can be used to derive apolynucleotide that encodes a polymerase polypeptide of the inventionand comprises preferred E. coli codons. The frequency of preferred codonusage exhibited by a host cell could be calculated by averagingfrequency of preferred codon usage in a large number of genes expressedby the host cell.

When synthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell. Thepercent deviation of the frequency of preferred codon usage for asynthetic gene from that employed by a host cell is calculated by firstdetermining the percent deviation of the frequency of usage of a singlecodon from that of the host followed by obtaining the average deviationover all codons.

DNA Binding Domains of the Present Invention

In some embodiments, the novel polymerases are conjugated to a DNAbinding domain. A DNA binding domain is a protein, or a defined regionof a protein, that binds to nucleic acid in a sequence-independentmatter, e.g., binding does not exhibit a gross preference for aparticular sequence. DNA binding domains may be single or doublestranded.

The DNA binding proteins are preferably thermostable. Examples of suchproteins include, but are not limited to, the Archaeal small basic DNAbinding proteins Sso7D and Sso7D-like proteins (see, e.g., Choli et al.,Biochimica et Biophysica Acta 950:193-203, 1988; Baumann et al.,Structural Biol. 1:808-819, 1994; and Gao et al, Nature Struc. Biol.5:782-786, 1998), Archaeal HMf-like proteins (see, e.g., Starich et al.,J. Molec. Biol. 255:187-203, 1996; Sandman et al., Gene 150:207-208,1994), and PCNA homologs (see, e.g., Cann et al., J. Bacteriology181:6591-6599, 1999; Shamoo and Steitz, Cell: 99, 155-166, 1999; DeFelice et al., J. Molec. Biol. 291, 47-57, 1999; and Zhang et al.,Biochemistry 34:10703-10712, 1995).

Sso7d and Sso7d-like proteins, Sac7d and Sac7d-like proteins, e.g.,Sac7a, Sac7b, Sac7d, and Sac7e are small (about 7,000 kd MW), basicchromosomal proteins from the hyperthermophilic archaebacteriaSulfolobus solfataricus and S. acidocaldarius, respectively. Theseproteins are lysine-rich and have high thermal, acid and chemicalstability. They bind DNA in a sequence-independent manner and whenbound, increase the T_(M) of DNA by up to 40° C. under some conditions(McAfee et al., Biochemistry 34:10063-10077, 1995). These proteins andtheir homologs are typically believed to be involved in stabilizinggenomic DNA at elevated temperatures. Suitable Sso7d-like DNA bindingdomains for use in the invention can be modified based on their sequencehomology to Sso7d. Typically, DNA binding domains that are identical toor substantially identical to a known DNA binding protein over acomparison window of about 25 amino acids, optionally about 50-100 aminoacids, or the length of the entire protein, can be used in theinvention. The sequence can be compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the described comparison algorithms or by manualalignment and visual inspection. For purposes of this patent, percentamino acid identity is determined by the default parameters of BLAST.

The HMf-like proteins are archaeal histones that share homology both inamino acid sequences and in structure with eukaryotic H4 histones, whichare thought to interact directly with DNA. The HMf family of proteinsform stable dimers in solution, and several HMf homologs have beenidentified from thermostable species (e.g., Methanothermus fervidus andPyrococcus strain GB-3a). The HMf family of proteins, once joined to TaqDNA polymerase or any DNA modifying enzyme with a low intrinsicprocessivity, can enhance the ability of the enzyme to slide along theDNA substrate and thus increase its processivity. For example, thedimeric HMf-like protein can be covalently linked to the N terminus ofTaq DNA polymerase, e.g., via chemical modification, and thus improvethe processivity of the polymerase.

Certain helix-hairpin-helix motifs have been shown to bind DNAnonspecifically and enhance the processivity of a DNA polymerase towhich it is fused (Pavlov et al., Proc Natl Acad Sci USA. 99:13510-5,2002).

Many but not all family B DNA polymerases interact with accessoryproteins to achieve highly processive DNA synthesis. A particularlyimportant class of accessory proteins is referred to as the slidingclamp. Several characterized sliding clamps exist as trimers insolution, and can form a ring-like structure with a central passagecapable of accommodating double-stranded DNA. The sliding clamp formsspecific interactions with the amino acids located at the C terminus ofparticular DNA polymerases, and tethers those polymerases to the DNAtemplate during replication. The sliding clamp in eukarya is referred toas the proliferating cell nuclear antigen (PCNA), while similar proteinsin other domains are often referred to as PCNA homologs. These homologshave marked structural similarity but limited sequence similarity.

Recently, PCNA homologs have been identified from thermophilic Archaea(e.g., Pyroccocus furiosus). Some family B polymerases in Archaea have aC terminus containing a consensus PCNA-interacting amino acid sequenceand are capable of using a PCNA homolog as a processivity factor (see,e.g., Cann et al., J. Bacteriol. 181:6591-6599, 1999 and De Felice etal., J. Mol. Biol. 291:47-57, 1999). These PCNA homologs are useful DNAbinding domains for the invention. For example, a consensusPCNA-interacting sequence can be joined to a polymerase that does notnaturally interact with a PCNA homolog, thereby allowing a PCNA homologto serve as a processivity factor for the polymerase. By way ofillustration, the PCNA-interacting sequence from Pyrococcus furiosusPolII (a heterodimeric DNA polymerase containing two family B-likepolypeptides) can be covalently joined to Pyrococcus furiosus PolI (amonomeric family B polymerase that does not normally interact with aPCNA homolog). The resulting fusion protein can then be allowed toassociate non-covalently with the Pyrococcus furiosus PCNA homolog togenerate a novel heterologous protein with increased processivityrelative to the unmodified Pyrococcus furiosus PolI.

Additional DNA binding domains suitable for use in the invention can beidentified by homology with known DNA binding proteins and/or byantibody crossreactivity, or may be found by means of a biochemicalassay. DNA binding domains may be synthesized or isolated using thetechniques described above.

Joining a DNA Binding Domain to a Polymerase

The DNA binding domain and the polymerase domain of the conjugate orfusion proteins of the invention can be joined by methods well known tothose of skill in the art. These methods include both chemical andrecombinant means.

Chemical means of joining a DNA binding protein to a polymerase domainare described, e.g., in Bioconjugate Techniques, Hermanson, Ed.,Academic Press (1996). These include, for example, derivitization forthe purpose of linking the two proteins to each other, either directlyor through a linking compound, by methods that are well known in the artof protein chemistry. For example, in one chemical conjugationembodiment, the means of linking the catalytic domain and the DNAbinding domain comprises a heterobifunctional-coupling reagent whichultimately contributes to formation of an intermolecular disulfide bondbetween the two moieties. Other types of coupling reagents that areuseful in this capacity for the present invention are described, forexample, in U.S. Pat. No. 4,545,985. Alternatively, an intermoleculardisulfide may conveniently be formed between cysteines in each moiety,which occur naturally or are inserted by genetic engineering. The meansof linking moieties may also use thioether linkages betweenheterobifunctional crosslinking reagents or specific low pH cleavablecrosslinkers or specific protease cleavable linkers or other cleavableor noncleavable chemical linkages.

The means of linking a DNA binding domain, e.g., Sso7d, and a polymerasedomain may also comprise a peptidyl bond formed between moieties thatare separately synthesized by standard peptide synthesis chemistry orrecombinant means. The conjugate protein itself can also be producedusing chemical methods to synthesize an amino acid sequence in whole orin part. For example, peptides can be synthesized by solid phasetechniques, such as, e.g., the Merrifield solid phase synthesis method,in which amino acids are sequentially added to a growing chain of aminoacids (see, Merrifield (1963) J. Am. Chem. Soc., 85:2149-2146).Equipment for automated synthesis of polypeptides is commerciallyavailable from suppliers such as PE Corp. (Foster City, Calif.), and maygenerally be operated according to the manufacturer's instructions. Thesynthesized peptides can then be cleaved from the resin, and purified,e.g., by preparative high performance liquid chromatography (seeCreighton, Proteins Structures and Molecular Principles, 50-60 (1983)).The composition of the synthetic polypeptides or of subfragments of thepolypeptide, may be confirmed by amino acid analysis or sequencing(e.g., the Edman degradation procedure; see Creighton, Proteins,Structures and Molecular Principles, pp. 34-49 (1983)).

In addition, nonclassical amino acids or chemical amino acid analogs canbe introduced as a substitution or addition into the sequence.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxy-proline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

In another embodiment, a DNA binding domain and polymerase domain can bejoined via a linking group. The linking group can be a chemicalcrosslinking agent, including, for example,succinimidyl-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Thelinking group can also be an additional amino acid sequence(s),including, for example, a polyalanine, polyglycine or similarly, linkinggroup.

In some embodiments, the coding sequences of each polypeptide in aresulting fusion protein are directly joined at their amino- orcarboxy-terminus via a peptide bond in any order. Alternatively, anamino acid linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Suchan amino acid linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Typical peptide linker sequences contain Gly, Ser,Val and Thr residues. Other near neutral amino acids, such as Ala canalso be used in the linker sequence. Amino acid sequences which may beusefully employed as linkers include those disclosed in Maratea et al.(1985) Gene 40:39-46; Murphy et al. (1986) Proc. Natl. Acad. Sci. USA83:8258-8262; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may generally be from 1 to about 50 amino acids in length,e.g., 3, 4, 6, or 10 amino acids in length, but can be 100 or 200 aminoacids in length. Linker sequences may not be required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

Other chemical linkers include carbohydrate linkers, lipid linkers,fatty acid linkers, polyether linkers, e.g., PEG, etc. For example,poly(ethylene glycol) linkers are available from Shearwater Polymers,Inc. Huntsville, Ala. These linkers optionally have amide linkages,sulfhydryl linkages, or heterobifunctional linkages.

Other methods of joining a DNA binding domain and polymerase domaininclude ionic binding by expressing negative and positive tails andindirect binding through antibodies and streptavidin-biotininteractions. (See, e.g., Bioconjugate Techniques, supra). The domainsmay also be joined together through an intermediate interactingsequence. For example, an Sso7D-interacting sequence, i.e., a sequencethat binds to Sso7D, can be joined to a polymerase. The resulting fusionprotein can then be allowed to associate non-covalently with the Sso7Dto generate an Sso7D-polymerase conjugate.

Production of Polypeptides Using Recombinant Techniques

As previously described, nucleic acids encoding the polymerase or DNAbinding domains can be obtained using routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994-1999).

In one example of obtaining a nucleic acid encoding a Sso7d domain usingPCR for use in the present invention, the nucleic acid sequence orsubsequence is PCR amplified, using a sense primer containing onerestriction site and an antisense primer containing another restrictionsite. This will produce a nucleic acid encoding the desired domainsequence or subsequence and having terminal restriction sites. Thisnucleic acid can then be easily ligated into a vector containing anucleic acid encoding a second domain, e.g., polymerase domain, andhaving the appropriate corresponding restriction sites. The domains canbe directly joined or may be separated by a linker, or other, proteinsequence. Suitable PCR primers can be determined by one of skill in theart using the sequence information provided in GenBank or other sources.Appropriate restriction sites can also be added to the nucleic acidencoding the protein or protein subsequence by site-directedmutagenesis. The plasmid containing the domain-encoding nucleotidesequence or subsequence is cleaved with the appropriate restrictionendonuclease and then ligated into an appropriate vector foramplification and/or expression according to standard methods.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods are described above and found in Berger,Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No.4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim& Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)3: 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell etal. (1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu andWallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117.

One of skill will recognize that modifications can additionally be madeto the polymerases of the present invention without diminishing theirbiological activity. Some modifications may be made to facilitate thecloning, expression, or incorporation of a domain into a fusion protein.Such modifications are well known to those of skill in the art andinclude, for example, the addition of codons at either terminus of thepolynucleotide that encodes the binding domain to provide, for example,a methionine added at the amino terminus to provide an initiation site,or additional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located restriction sites or termination codons orpurification sequences.

One or more of the domains may also be modified to facilitate thelinkage of a variant polymerase domain and DNA binding domain to obtainthe polynucleotides that encode the fusion polymerases of the invention.Thus, DNA binding domains and polymerase domains that are modified bysuch methods are also part of the invention. For example, a codon for acysteine residue can be placed at either end of a domain so that thedomain can be linked by, for example, a sulfide linkage. Themodification can be performed using either recombinant or chemicalmethods (see, e.g., Pierce Chemical Co. catalog, Rockford Ill.).

The DNA binding and polymerase domains comprised by a recombinant fusionprotein are often joined by linker domains, usually polypeptidesequences including Gly, Ser, Ala, and Val such as those describedabove. In some embodiments, proline residues are incorporated into thelinker to prevent the formation of significant secondary structuralelements by the linker.

Expression Cassettes and Host Cells for Expressing Polypeptides

The polymerases of the present invention can be expressed in a varietyof host cells, including E. coli, other bacterial hosts, yeasts,filamentous fungi, and various higher eukaryotic cells such as the COS,CHO and HeLa cells lines and myeloma cell lines. Techniques for geneexpression in microorganisms are described in, for example, Smith, GeneExpression in Recombinant Microorganisms (Bioprocess Technology, Vol.22), Marcel Dekker, 1994. Examples of bacteria that are useful forexpression include, but are not limited to, Escherichia, Enterobacter,Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus,Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.Filamentous fungi that are useful as expression hosts include, forexample, the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, andPyricularia. See, e.g., U.S. Pat. No. 5,679,543 and Stahl and Tudzynski,Eds., Molecular Biology in Filamentous Fungi, John Wiley & Sons, 1992.Synthesis of heterologous proteins in yeast is well known and describedin the literature. Methods in Yeast Genetics, Sherman, F., et al., ColdSpring Harbor Laboratory, (1982) is a well recognized work describingthe various methods available to produce the enzymes in yeast.

There are many expression systems for producing the polymerasepolypeptides of the present invention that are well know to those ofordinary skill in the art. (See, e.g., Gene Expression Systems,Fernandex and Hoeffler, Eds. Academic Press, 1999; Sambrook $ Russell,supra; and Ausubel et al, supra.) Typically, the polynucleotide thatencodes the variant polypeptide is placed under the control of apromoter that is functional in the desired host cell. An extremely widevariety of promoters are available, and can be used in the expressionvectors of the invention, depending on the particular application.Ordinarily, the promoter selected depends upon the cell in which thepromoter is to be active. Other expression control sequences such asribosome binding sites, transcription termination sites and the like arealso optionally included. Constructs that include one or more of thesecontrol sequences are termed “expression cassettes.” Accordingly, thenucleic acids that encode the joined polypeptides are incorporated forhigh level expression in a desired host cell.

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21-25); and the lambda-derived P_(L) promoter andN-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128).The particular promoter system is not critical to the invention, anyavailable promoter that functions in prokaryotes can be used. Standardbacterial expression vectors include plasmids such as pBR322-basedplasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, λ-phage derived vectors, andfusion expression systems such as GST and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc, HA-tag, 6-His (SEQ ID NO:47) tag, maltosebinding protein, VSV-G tag, anti-DYKDDDDK (SEQ ID NO:48) tag, or anysuch tag, a large number of which are well known to those of skill inthe art.

For expression of in prokaryotic cells other than E. coli, a promoterthat functions in the particular prokaryotic species is required. Suchpromoters can be obtained from genes that have been cloned from thespecies, or heterologous promoters can be used. For example, the hybridtrp-lac promoter functions in Bacillus sp. in addition to E. coli. Theseand other suitable bacterial promoters are well known in the art and aredescribed, e.g., in Sambrook et al. and Ausubel et al. Bacterialexpression systems for expressing the proteins of the invention areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kitsfor such expression systems are commercially available.

Eukaryotic expression systems for mammalian cells, yeast, and insectcells are well known in the art and are also commercially available. Inyeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and YeastReplicating plasmids (the YRp series plasmids) and pGPD-2. Expressionvectors containing regulatory elements from eukaryotic viruses aretypically used in eukaryotic expression vectors, e.g., SV40 vectors,papilloma virus vectors, and vectors derived from Epstein-Barr virus.Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the CMV promoter, SV40 earlypromoter, SV40 later promoter, metallothionein promoter, murine mammarytumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter,or other promoters shown effective for expression in eukaryotic cells.

Either constitutive or regulated promoters can be used in the presentinvention. Regulated promoters can be advantageous because the hostcells can be grown to high densities before expression of the fusionpolypeptides is induced. High level expression of heterologous proteinsslows cell growth in some situations. An inducible promoter is apromoter that directs expression of a gene where the level of expressionis alterable by environmental or developmental factors such as, forexample, temperature, pH, anaerobic or aerobic conditions, light,transcription factors and chemicals.

For E. coli and other bacterial host cells, inducible promoters areknown to those of skill in the art. These include, for example, the lacpromoter, the bacteriophage lambda P_(L) promoter, the hybrid trp-lacpromoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc.Nat'l. Acad. Sci. USA 80: 21), and the bacteriophage T7 promoter(Studier et al. (1986) J. Mol. Biol.; Tabor et al. (1985) Proc. Nat'l.Acad. Sci. USA 82: 1074-8). These promoters and their use are discussedin Sambrook et al., supra.

Inducible promoters for other organisms are also well known to those ofskill in the art. These include, for example, the metallothioneinpromoter, the heat shock promoter, as well as many others.

Translational coupling may be used to enhance expression. The strategyuses a short upstream open reading frame derived from a highly expressedgene native to the translational system, which is placed downstream ofthe promoter, and a ribosome binding site followed after a few aminoacid codons by a termination codon. Just prior to the termination codonis a second ribosome binding site, and following the termination codonis a start codon for the initiation of translation. The system dissolvessecondary structure in the RNA, allowing for the efficient initiation oftranslation. See Squires, et. al. (1988), J. Biol. Chem. 263:16297-16302.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in bacteria. Such vectors are commonly usedin the art. A plethora of kits are commercially available for thepurification of plasmids from bacteria (for example, EasyPrepJ,FlexiPrepJ, from Pharmacia Biotech; StrataCleanJ, from Stratagene; and,QIAexpress Expression System, Qiagen). The isolated and purifiedplasmids can then be further manipulated to produce other plasmids, andused to transform cells.

The polypeptides of the present invention can be expressedintracellularly, or can be secreted from the cell. Intracellularexpression often results in high yields. If necessary, the amount ofsoluble, active fusion polypeptide may be increased by performingrefolding procedures (see, e.g., Sambrook et al., supra.; Marston etal., Bio/Technology (1984) 2: 800; Schoner et al., Bio/Technology (1985)3: 151). Polypeptides of the invention can be expressed in a variety ofhost cells, including E. coli, other bacterial hosts, yeast, and varioushigher eukaryotic cells such as the COS, CHO and HeLa cells lines andmyeloma cell lines. The host cells can be mammalian cells, insect cells,or microorganisms, such as, for example, yeast cells, bacterial cells,or fungal cells.

Once expressed, the polypeptides can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, gel electrophoresis and thelike (see, generally, R. Scopes, Protein Purification, Springer-Verlag,N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification., Academic Press, Inc. N.Y. (1990)). Substantially purecompositions of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity are most preferred. Once purified,partially or to homogeneity as desired, the polypeptides may then beused (e.g., as immunogens for antibody production).

To facilitate purification of the polypeptides of the invention, thenucleic acids that encode the polypeptides can also include a codingsequence for an epitope or “tag” for which an affinity binding reagentis available. Examples of suitable epitopes include the myc and V-5reporter genes; expression vectors useful for recombinant production offusion polypeptides having these epitopes are commercially available(e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His andpcDNA3.1/V5-His are suitable for expression in mammalian cells).Additional expression vectors suitable for attaching a tag to the fusionproteins of the invention, and corresponding detection systems are knownto those of skill in the art, and several are commercially available(e.g., FLAG” (Kodak, Rochester N.Y.). Another example of a suitable tagis a polyhistidine sequence, which is capable of binding to metalchelate affinity ligands. Typically, six adjacent histidines (SEQ IDNO:47) are used, although one can use more or less than six. Suitablemetal chelate affinity ligands that can serve as the binding moiety fora polyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli, E.(1990) “Purification of recombinant proteins with metal chelatingadsorbents” In Genetic Engineering: Principles and Methods, J. K.Setlow, Ed., Plenum Press, NY; commercially available from Qiagen (SantaClarita, Calif.)).

One of skill in the art would recognize that after biological expressionor purification, the polymerase peptide (s) may possess a conformationsubstantially different than the native conformations of the constituentpolypeptides. In this case, it may be necessary or desirable to denatureand reduce the polypeptide and then to cause the polypeptide to re-foldinto the preferred conformation. Methods of reducing and denaturingproteins and inducing re-folding are well known to those of skill in theart (See, Debinski et al. (1993) J. Biol. Chem. 268: 14065-14070;Kreitman and Pastan (1993) Bioconjug. Chem. 4: 581-585; and Buchner etal. (1992) Anal. Biochem. 205: 263-270). Debinski et al., for example,describe the denaturation and reduction of inclusion body proteins inguanidine-DTE. The protein is then refolded in a redox buffer containingoxidized glutathione and L-arginine.

Assays to Evaluate Polymerase Activity

Activity of a polymerase can be measured using a variety of assays thatcan be used to determine processivity or modification activity of apolymerase. Improvement in activity may include both increasedprocessivity and increased efficiency.

The polymerases of the present invention, e.g. SEQ ID NO:2 and SEQ IDNO:4, exhibit polymerase activity, e.g., processivity, primer/templatebinding specificity, and 3′ to 5′ exonuclease activity. The activitiescan be measured using techniques that are standard in the art.

For example, polymerase processivity can be measured by a variety ofmethods known to those of ordinary skill in the art. Polymeraseprocessivity is generally defined as the number of nucleotidesincorporated during a single binding event of a modifying enzyme to aprimed template. For example, a 5′ FAM-labeled primer is annealed tocircular or linearized ssM13 mp18 DNA to form a primed template. Inmeasuring processivity, the primed template usually is present insignificant molar excess to the polymerase so that the chance of anyprimed template being extended more than once by the polymerase isminimized. The primed template is therefore mixed with the polymerase ata ratio such as approximately 4000:1 (primed DNA:DNA polymerase) in thepresence of buffer and dNTPs. MgCl₂ is added to initiate DNA synthesis.Samples are quenched at various times after initiation, and analyzed ona sequencing gel. At a polymerase concentration where the median productlength does not change with time or polymerase concentration, the lengthcorresponds to the processivity of the enzyme. The processivity of aprotein of the invention, e.g., SEQ ID NO:2 or SEQ ID NO:4, is thencompared to the processivity of a wild type enzyme.

Efficiency can be demonstrated by measuring the ability of an enzyme toproduce product. Increased efficiency can be demonstrated by measuringthe increased ability of an enzyme to produce product. Such an analysismeasures the stability of the double-stranded nucleic acid duplexindirectly by determining the amount of product obtained in a reaction.For example, a PCR assay can be used to measure the amount of PCRproduct obtained with a short, e.g., 12 nucleotide in length, primerannealed at an elevated temperature, e.g., 50° C. In this analysis,enhanced efficiency is shown by the ability of a polymerase to producemore product in a PCR reaction using the 12 nucleotide primer annealedat 50° C.

Efficiency can also be measured, e.g., in a real-time PCR. The Ct valuerepresents the number of cycles required to generate a detectable amountof DNA (a “detectable” amount of DNA is typically 2×, usually 5×, 10×,100× or more above background). An efficient polymerase may be able toproduce a detectable amount of DNA in a smaller number of cycles by moreclosely approaching the theoretical maximum amplification efficiency ofPCR. Accordingly, a lower Ct value reflects a greater amplificationefficiency for the enzyme.

Long PCR may be used as another method of demonstrating enhancedefficiency. For example, an enzyme with enhanced efficiency typicallyallows the amplification of a long amplicon (>5 kb) in a shorterextension time compared to an enzyme with relatively lower efficiency.

Assays such as salt sensitivity can also be used to demonstrateimprovement in efficiency or equivalent efficiency of a polymerase ofthe invention. A polymerase of the present invention may exhibitincreased tolerance to high salt concentrations, i.e., a processiveenzyme with increased processivity can produce more product in highersalt concentrations. For example, a PCR analysis can be performed todetermine the amount of product obtained in a reaction using apolymerase of the present invention compared to a wild type polymerasein reaction conditions with high salt, e.g., 80 mM.

Other methods of assessing efficiency of the polymerases of theinvention can be determined by those of ordinary skill in the art usingstandard assays of the enzymatic activity of a given modificationenzyme.

Primer/template specificity is the ability of an enzyme to discriminatebetween matched primer/template duplexes and mismatched primer/templateduplexes. Specificity can be determined, for example, by comparing therelative yield of two reactions, one of which employs a matched primer,and one of which employs a mismatched primer. An enzyme with increaseddiscrimination will have a higher relative yield with the matched primerthan with the mismatched primer, i.e., the ratio of the yield in thereaction using the matched primer vs. the reaction using the mismatchedprimer is about 1 or above. This ratio can then be compared to the yieldobtained in a parallel set of reactions employing a wild typepolymerase.

In other assays for improvement, the exonuclease activity of apolymerase can also be measured, as described in the “Examples” section.In some instances, desired improvements may take into account multiplefunctions of a polymerase. For example, one may want to tailor the ratioof exonuclease activity to polymerization activity.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

EXAMPLES Example 1 Generation of Hybrid Polymerases

Pfu polymerase is a commercially available (Stratagene, La Jolla,Calif.) family B DNA polymerase isolated from Pyrococcus furiosus. DeepVent® is a commercially available (New England Biolabs, Beverly, Mass.)family B DNA polymerase isolated from Pyrococcus sp. GB-D. Being 775amino acids in length, these proteins are twice as large as a typicalprotein. They share a variety of activities including DNA binding,nucleotide binding, nucleotide addition, pyrophosphorolysis, and 3′ to5′ exonuclease (proofreading) activities. The method of generating ahybrid polymerase can be applied to any one of the activities encoded bythese large proteins by being applied to one domain of the protein. Inthis example, the method was applied to each of the different enzymaticactivities, by making a hybrid library for the entire protein.

The protein sequences of Pfu polymerase and Deep Vent® polymerase werealigned. The alignment and a consensus hybrid protein sequence, in whichX indicates the residues at which the parents differ, are shown inFIG. 1. The amino acid sequences of Pfu and Deep Vent® differ from oneanother at 115 locations. The sequences are 85% identical over thecomplete sequence. One 18-amino-acid-region is only 56% identical.Hybrid Deep Vent®/Pfu proteins were produced by creating a collection ofoligonucleotides that encodes a blend of sequences from the two parentsand then assembling the oligonucleotides in a library of full-lengthpolymerase proteins.

As stated, the alignment found 115 differences between the Pfu and DeepVent® amino acid sequences. An E. coli codon usage table was then usedto compare the various codons that can encode the amino acids and deducean minimal encoding sequence. In many instances, a single nucleic aciddegeneracy could encode both amino acids. For example, the parentproteins differ at amino acid position 15 where Pfu has a valine (Val)and Deep Vent® an isoleucine (Ile). It is possible to encode Val usingGTT and Ile using ATT. The oligonucleotide synthesis machine wastherefore programmed to produce product with half G and half A atnucleotide position 43 of the protein-coding DNA. Thus, a codon with aRTT where either a G or an A is introduced into the first nucleotideposition of the codon will provide a pool of oligonucleotides, some ofwhich have a GTT at that position; the others of which have an ATT atthat position.

In the alignment of Pfu and Deep Vent®, 98 of the 115 differences couldbe simply incorporated into the library by introducing a singledegeneracy at one nucleotide residue of the codon that encoded thedifferent amino acids.

The remaining 17 differences required two nucleotides to be changed inorder to encode the two parental sequences. These changes forced thepossibility that two non-parental amino acid sequences would exist inthe resulting library. An example of this is residue 72, at which Pfuhas a glutamate (Glu) and the Deep Vent® has an arginine (Arg). Glu isencoded by GAR and Arg by CGN or AGR. The minimal encoding sequence(A/G)(A/G)G was selected to potentially encode the parent sequences atposition 214 through 216 of the hybrid protein-coding region. Thiscombination will also generate nucleotides encoding glycine (GGG) andlysine (AAG). This situation was determined to be tolerable even thoughglycine is not similar to either parental amino acid because suchsituations were rare relative to the size of the protein.

Incorporation of a potential stop codon at amino acid residue 758(nucleic acid residues 2272 and 2273) was also deemed to be tolerable.This stop codon made ¼ of the library useless. Amino acid residue 566(nucleotides 1696 through 1698) was made a lysine by mistake; it shouldhave contained a nucleotide degeneracy that encoded lysine or asparticacid.

For each strand of the minimal encoding sequence, a set of degenerateoligonucleotides of approximately 100 bases in length, and separated bygaps of 40 bases, was synthesized. The oligonucleotide sequences on thetwo strands were arranged so that the oligonucleotides from the firststrand spanned the gaps on the second strand and overlapped theoligonucleotides of the second strand by 30 bases (FIG. 2). Thisoligonucleotide set was used in assembly PCR as follows. Overlappingoligonucleotides were paired, annealed to each other, and extended usinga thermostable high fidelity polymerase. High concentrations ofoligonucleotide and a minimal number of thermal cycles (no more than 5)were used. The products of the first cycle were double-strandedfragments of approximately 170 base pairs in length. These fragmentswere band-purified from a gel and used for the next cycle of pairing andprimer extension to generate a new double-stranded fragment of about 310base pairs in length. This cycle was repeated until the entire sequencewas obtained as a collection of fragments of about 500 bases in length.At this point, particular fragments were selected and sequenced toassess the integrity of the procedure. It was found that theoligonucleotides purchased were of low quality, resulting in excessiveunintended mutations. A number of segments containing no unintendedmutations were chosen and used to assemble full-length genes usingrestriction sites that had been incorporated at the ends of eachfragment and conventional molecular biology techniques. Four full-lengthclones were assembled and the encoded proteins were expressed in pET11(Novogene, Madison, Wis.). Expression by all four clones was confirmedby SDS-PAGE. These clones were names Hyb1 to Hyb4.

A second collection of libraries was constructed on a custom basis byBlue Heron Biotechnology (Bothell, Wash.) using “Genemaker” technology.The complete coding sequence was delivered as four fragment librariesthat could be assembled into a full-length hybrid genes. Two full-lengthassembled clones were obtained and sequenced to verify validity of thelibrary. These clones were named Phy1 and Phy2. Clones from this librarycontained only proper hybrid sequences including the degeneracies atposition 566 (lysine/aspartic acid) and 758 (tyrosine/tryptophan)discussed earlier. The full-length sequences were cloned into expressionvectors and protein of the expected size were produced.

Hybrid polymerase protein was expressed and purified from each of thesix clones from the two libraries. Purification was performed asfollows.

Purification of Hybrid Polymerases

This section describes methodology for isolating a hybrid polymerase.Following induction of expression in E. coli, the cells were centrifugedand the pellets stored at −20° C. to −80° C. One milliliter of Buffer A(Buffer: 50 mM Tris (8.0); 50 mM Dextrose; 1 mM EDTA) was added forevery 100 ml of starting culture and the cells were lysed with 4 mg/mlof powdered lysozyme at 72° C. MgCl₂ and CaCl₂ were added to aconcentration of 2 mM, followed by the addition of 1 unit/ml of DNase I.The sample was shaken slowly for 10 min at room temperature. One ml ofBuffer B (10 mM Tris (8.0); 50 mM KCl; 1 mM EDTA; 0.5% Tween 20; 0.5%NP40) was added per 100 ml starting culture and the sample then shakenslowly at room temperature for 15 min. The sample was transferred to acentrifuge tube and incubated at 72° C. for 1 hour followed bycentrifugation at 4000×g at 4° C. for 15 min. The supernatant wascollected and 0.476 gm/ml of (NH₄)₂SO₄ was added and the sample wasmixed slowly at 4° C. for 1 hour and then centrifuged at 15,000×g at 4°C. for 15 min.

The pellet was resuspended in, and dialyzed against HiTrap Q ‘A’ Buffer(20 mM Tris (7.9); 50 mM NaCl; 5 mM β-mercaptoethanol). The suspensionwas then loaded onto a ÄKTAprime HiTrap Q chromatography column(Amersham Biosciences) equilibrated and run using method #2 per themanufacturers instructions using HiTrap Q buffers ‘A’ and ‘B’ (‘A’buffer with 1 M NaCl). Fractions containing the polymerase were combinedand dialyzed against P-1 Loading Buffer (20 mM Tris (7.9); 50 mM NaCl).The sample was bound to a liquid chromatography column of P-11 resin(Amersham Biosciences), washed with P-11 Buffer ‘B’ (20 mM Tris (7.9);150 mM NaCl), then eluted using P-11 Elution Buffer (20 mM Tris (7.9);400 mM NaCl). The eluted fractions were dialyzed against HiTrap SP ‘A’buffer (20 mM Tris (6.8); 50 mM NaCl; 5 mM β-mercaptoethanol) theninjected onto a ÄKTAprime HiTrap SP chromatography column equilibratedand run using method #2 per the manufacturers instructions using HiTrapSP ‘A’ and ‘B’ Buffer (‘A’ buffer with 1 M NaCl). Fractions containingPhS1 were concentrated using a YM-30 Centricon protein concentrator(Millipore). The sample was then dialyzed against buffer containing 50mM Tris (pH 8.2); 0.1 mM EDTA; 1 mM DTT; 0.1% NP40; 0.1% Tween 20. Thefinal volume was then measured and 1.47×85% glycerol, and 0.015×10%NP-40 and 10% Tween 20 added. The sample was stored at −20° C.

Of the six hybrid polymerase proteins generated from the two libraries,all had DNA polymerase activity.

Sso7d fusion polymerases (see, e.g., WO0192501) were prepared using someof the hybrid polymerase proteins and compared to the parental Pfupolymerase with and without Sso7d (designated as “Pfu” and “PfS”,respectively) in exonuclease assays and extension assays. Sso7d fusionsof Hyb clones are designated HyS; Sso7d fusions of the Phy clones aredesignated PhS. The most thoroughly studied hybrid protein was PhS1.

To measure exonuclease activity, a 45 base long primer with thefollowing sequence was synthesized:5′-FAM-TTTTTTGAGGTGTGTCCTACACAGCGGAGTGTAGGA CACACCTCT* 3′ (SEQ IDNO:49), wherein T*=is an amino-link dT with the quencher, DAB (dabcyl)attached. The sequence forms a 16 base pair stem loop structure with aT:T* mismatch at the quencher-labeled base. The 5′ unbase-paired poly Tsequence keeps FAM (6 carboxy-fluorescein) in close proximity to thequenching dye so the FAM, if excited, it will not fluoresce.

The oligonucleotide was combined with buffer and the enzyme andincubated in a real time detection instrument, the DNA Engine OpticonSystem (MJ Research, Inc.). This instrument excites the FAM and detectsany fluorescence if present. In the absence of 3′ to 5′ exonucleaseactivity, there is only background fluorescence because FAM is quenchedby DAB. However if the enzyme does have 3′ to 5′ exonuclease activity,the T:T* mismatch is recognized and the 3′-T* is removed. The DAB isreleased and will no longer quench the FAM fluorescence. The OpticonSystem will detect the increase in fluorescence with increasing time(readings were taken every 10 sec at 65° C.). The rate of fluorescenceincrease directly reflects the amount of 3′ to 5′ exonuclease activity.An increase in fluorescence greater than control levels shows that theenzyme has 3′ to 5′ exonuclease activity. The results (FIG. 3) of thisanalysis are discussed below.

FIG. 4 shows results of a comparison of a hybrid and a parent polymerasein extension assays. Even with excess enzyme (80 U/ml), Pfu could notamplify any amplicon longer than 2 kb. An Sso7d fusion to Pfu polymerase(PfS) amplified a 10 kb fragment given a 1 min extension time. PhS1amplified a 15 kb fragment (arrow) in 80 mM KCl with a 1 minuteextension time. Further, PhS1 was also able to perform long PCR under avariety of salt conditions.

Characterization of Additional Hybrid Polymerases

Five additional hybrid clones were isolated from the second librarydirectly as Sso7d fusions and were designated PhS3 to PhS7. Thepolymerases were tested for polymerase and exonuclease activity. Table 1summarizes characteristics of the various hybrid proteins analyzed inthis example. PhS2 has two mutations at sites other than a target site.PhS3 is truncated due to an early stop codon. PhS4 has one deletion andone mutation. The “Hyb” and “HyS” polymerases also comprise mutations atpositions other than the target sites, probably due to faultyoligonucleotide synthesis.

TABLE 1 Number Number Pfu D. vent Relative Temp. parent parent specificPol Activity Full-length KCL Opt Stab. Processivity residues residuesactivity PhS1 Yes Yes 80-100 mM  3 hr, 97.5 26-30 55 60   1.5 PhS2 YesYes 160-180 mM  3 hr+, 24-28 64 51 4 97.5 PhS3 No No N/A N/A N/A N/A N/An.d. PhS4 No No; minus N/A N/A N/A 56 58 n.d. one Pfu/DV amino acid PhS5Yes Yes 40-80 mM 3 hr, 97.5 nd 52 63 1 Ph 6 No No N/A N/A N/A 55 60 n.d.Ph 7 Yes Yes 40-80 mM 3 hr, 97.5 nd 54 61 2 Hyb1 Yes Yes nd 10 min* 2-4nt 59 46 n.d. HyS1 Yes Yes 90-100 mM  8-14 min*  11 nt 59 46 2 Hyb2**Yes No nd n.d. n.d. 50 53 n.d. Hyb3** Yes No nd n.d. n.d. 51 47 n.d.HyS4 Yes Yes 80-90 mM <1 min* n.d. 51 50 n.d. All polymerases designated“PhS” are Sso7d fusions. “HyS1” is Hyb1 with Sso7d at the C-terminus.“HyS4” has Sso7d at the C-terminus.

The exonuclease activity of various hybrid polymerases was alsoevaluated as described above. The polymerase- to -3′-exonuclease ratiosfor several commercially available enzymes, including the parentalproteins and isolates from the hybrid library, were compared. DyNAzymeEXT, an enzyme used in long accurate PCR, is a blend of a Family Bpolymerase with 3′ to 5′ exonuclease activity, and a Family A polymerasethat lacks any proofreading activity. Too much exonuclease activity isdetrimental because it digests primers instead of extending them. Pfuand Deep Vent® are the parental Family B polymerases which both havehigh exonuclease activity. PfS (a Pfu-Sso7d fusion enzyme) has increasedpolymerase activity. HyS1, PhS1, PhS2, PhS5, and PhS7 are isolates fromthe hybrid libraries. Surprisingly, the results (FIG. 3) show that thehybrid proteins vary greatly in their polymerase to exonucleaseactivities, both relative to the parent proteins and each other. PhS1has a polymerase to exonuclease activity ratio approaching that of theenzyme blend.

These results show that multiple polymerase hybrid isolates from twodifferent libraries were active. Furthermore, the example shows that themethod also allows for generating hybrids for different domains, i.e.,polymerase activity domain vs. exonuclease activity domain.

A comparison of the sequences of the parent and various hybrid proteinsis presented in FIG. 5. As can be seen, a signature sequence, i.e., aninvariable sequence element, is present in all of the proteins. Thiselement (FIG. 6) contains the nucleotide binding motif and ischaracteristic of Pfu/DeepVent polymerases generated using the methoddescribed herein. The sites that differ between the parent polymerasesare indicated.

Example 2 Substantially Identical Polymerase Gene Synthesis

The following is a preferred method of generating polymerase nucleicacids encoding polymerases substantially identical to a polymerase ofthe invention, e.g., SEQ ID NO:2 or SEQ ID NO:4. A set of conservativesubstitutions are chosen. A degenerate sequence is constructed, wherethe degenerate positions in the nucleotide encode, in their alternativeforms, at least the two amino acids corresponding to the wild-type aminoacid and the conservative substitution. For each strand of thedegenerate sequence, a set of degenerate oligonucleotides ofapproximately 100 bases in length, and separated by gaps of 40 bases, issynthesized. The oligonucleotide sequences on the two strands arearranged so that the oligonucleotides from the first strand span thegaps on the second strand and overlap the oligonucleotides of the secondstrand by 30 bases. This oligonucleotide set is used in assembly PCR asfollows. Overlapping oligonucleotides are paired, annealed to eachother, and extended using a thermostable high fidelity polymerase. Highconcentrations of oligonucleotide and a minimal number of thermal cycles(no more than 5) are used whenever possible. The products of the firstcycle are double-stranded fragments of length approximately 170 bases.These are band-purified from a gel and used for the next cycle ofpairing and primer extension to generate new double-stranded fragmentsof length approximately 310 bases. This cycle is repeated until theentire sequence has been obtained in a single fragment. If at any pointthe quantity of the product becomes too low, the amount can be increasedby PCR using short (15-30) base primers corresponding to the ends ofparticular desired fragments. Cloning of partial gene sequences, and/orcutting with restriction enzymes and ligating subfragments together, areadditional techniques that may be used to improve the efficiency of thegene construction process. When the entire gene is synthesized, it iscloned into a vector suitable for protein expression. Because thesequence is degenerate, cloning will produce a library of related butdifferent clones, which must be screened to eliminate those clones thatdo not produce a functional protein or which are not substantiallyidentical to the target polymerase.

TABLE OF POLYMERASE SEQUENCES SEQ ID NO: 1 Phy1 nucleic acid sequenceATGATCCTGGATGCTGACTACATCACTGAAGAAGGCAAACCGGTTATCCGTCTGTTCAAAAAAGAGAACGGCGAATTTAAGATTGAGCATGATCGCACCTTTCGTCCATACATTTACGCTCTGCTGAAAGATGATTCTAAGATTGAGGAAGTTAAAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGCGGAAAAGGTAGAAAAGAAATTTCTGGGCAGACCAATCACCGTGTGGAGACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGAGAAAATTCGCGAACATTCTGCAGTTGTTGACATCTTCGAATACGATATTCCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGATGAAGAACTCAAGCTCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAGAAGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAGCGCTTTCTCAAAATTATCCGCGAGAAGGATCCGGACATTATCATTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGACTATCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTATCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTCGTCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAAAGGCGTGGGAAACCGGTGAGGGCCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCTTCCCAATGGAAGCTCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTTTCTCCTGCGCAAAGCGTACGAACGCAACGAACTGGCTCCGAACAAGCCAGATGAACGTGAGTATGAACGCCGTCTCCGCGAGTCTTACGCTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAACATCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAGAAACTATGATGTTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCCGGGCTTTATTCCGTCTCTCCTGAAACGTCTGCTCGATGAACGCCAAAAGATTAAGACTAAAATGAAGGCGTCCCAGGATCCGATTGAAAAAATAATGCTCGACTATCGCCAAAGAGCGATTAAAATCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTAAGTCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGGATTACATTAACGCGAAGCTCCCGGGTCTCCTGGAGCTCGAATATGAAGGCTTTTATAAACGCGGCTTCTTCGTTACCAAGAAGAAATATGCGCTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAGAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAGAATTGTAAAAGAAGTAACCCAAAAGCTCTCTAAATATGAAATTCCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAGACTGGCTGCTAAAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATACGATCCGAGAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGCTGGCAAAAGACTAAACAGACTGGCCTCACTTCTTGGCTCAACATTAAAAAATCC SEQ ID NO: 2 Phy1 polypeptide sequenceMILDADYITEEGRPVIRLFKKENGEFKIEHDRTFRPYIYALLKDDSKIEEVKKITAERHGKIVRIVDAEKVEKKFLGRPITVWRLYFEHPQDVPTIIREKIREHSAVVDWEYDTPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFGKGPIIMISYADEEEAKVITWKKIDLPYVEVVSSEREMIKRFLKIIREKDPDIIITYNGDSFDLPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVIRRTTNLPTYTLEAVYEAIFGKPKEKVYADEIAKAWETGEGLERVAKYSMEDAKATYELGKEFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEREYERRLRESYAGGFVKEPEKGLWENIVSLDFRALYPSIIITHNVSPDTLNREGCRNYDVAPEVGHKFCKDFPGFIPSLLKRLLDERQKIKTKMKASQDPIEKIMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVWKELEEKFGFKVLYIDTDGLYATIPGGKSEEIKKKALEFVDYINAKLPGLLELEYEGFYKRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVRIVKEVTQKLSKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKRLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPRKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRWQKTKQTGLTSWLNIIKKS SEQ ID NO: 3 Nucleic acid sequence encodingPhS1, a fusion protein comprising Phy1 and SSo7d, with the linker andthe Sso7d coding region in lower case, and the linker region in bold.ATGATCCTGGATGCTGACTACATCACTGAAGAAGGCAAACCGGTTATCCGTCTGTTCAAAAAAGAGAACGGCGAATTTAAGATTGAGCATGATCGCACCTTTCGTCCATACATTTACGCTCTGCTGAAAGATGATTCTAAGATTGAGGAAGTTAAAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGCGGAAAAGGTAGAAAGAAATTTCTGGGCAGACCAATCACCGTGTGGAGACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGAGAAAATTCGCGAACATTCTGCAGTTGTTGACATCTTCGAATACGATATTCCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGATGAAGAACTCAAGCTCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAGAAGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAGCGCTTTCTCAAAATTATCCGCGAGAAGGATCCGGACATTATCATTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGACTATCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTATCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTCGTCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAAAGGCGTGGGAAACCGGTGAGGGCCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCTTCCCAATGGAAGCTCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTTTCTCCTGCGCAAAGCGTACGAACGCAACGAACTGGCTCCGAACAAGCCAGATGAACGTGAGTATGAACGCCGTCTCCGCGAGTCTTACGCTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAACATCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAGAAACTATGATGTTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCCGGGCTTTATTCCGTCTCTCCTGAAACGTCTGCTCGATGAACGCCAAAAGATTAAGACTAAAATGAAGGCGTCCCAGGATCCGATTGAAAAAATAATGCTCGACTATCGCCAAAGAGCGATTAAAATCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTAAGTCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGGATTACATTAACGCGAAGCTCCCGGGTCTCCTGGAGCTCGAATATGAAGGCTTTTATAAACGCGGCTTCTTCGTTACCAAGAAGAAATATGCGCTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAGAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAGAATTGTAAAAGAAGTAACCCAAAAGCTCTCTAAATATGAAATTCCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAGACTGGCTGCTAAAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATACGATCCGAGAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGCTGGCAAAAGACTAAACAGACTGGCCTCACTTCTTGGCTCAACATTAAAAAATCCggtaccggcggtggcggtgcaaccgtaaagttcaagtacaaaggcgaagaaaaagaggtagacatctccaagatcaagaaagtatggcgtgtgggcaagatgatctccttcacctacgacgagggcggtggcaagaccggccgtggtgcggtaagcgaaaaggacgcgccgaaggagctgctgcagatgctggagaagcagaaaaagtga SEQ ID NO: 4 The amino acid sequenceof PhS1 (a PHY-SSo7d fusion protein), with the linker and the Sso7dcoding region in lower case, and the linker region in bold.MILDADYITEEGKPVIRLFKKENGEFKIEHDRTFRPYIYALLKDDSKIEEVKKITAERHGKIVRIVDAEKVEKKFLGRPITVWRLYFEHPQDVPTIREKIREHSAVVDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFGKGPIIMISYADEEEAKVITEKKIDLPYVEVVSSEREMIKRFLKIIREKDPDIIITYNGDSFDLPYLAKRAEKLGIIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWETGEGLERVAKYSMEDAKATYELGKEFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEREYERRLRESYAGGFVKEPEKGLWENIVSLDFRALYPSIIITHNVSPDTLNREGCRNYDVAPEVGHKFCKDFPGFIPSLLKRLLDERQKIKTKMKASQDPIEKIMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVWKELEEKEGFKVLYIDTDGLYATIPGGKSEEIKKKALEFVDYINAKLPGLLELEYEGFYKRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVRIVKEVTQKLSKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVARRLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPRKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRWQKTKQTGLTSWLNIKKSgtggggatvkfkykgeekevdiskikkvwrvgkmisftydegggktgrgavsekdapkellqmlekqkk* SEQ ID NO: 5 PhS2 nucleicacid sequence ATGATCCTGGATGTTGACTACATCACTGAAGAAGGCAAACCGGTTATCCGTCTGTTCAAAAAAGAGAACGGCGAATTTAAGGTTGAGTATGATCGCACCTTTCGTCCATACATTTACGCTCTGCTGAAAGATGATTCTAAGATTGATGAAGTTAGAAAAATCACTGGTGAGCGCCATGGCAAGATTGTTCGTATCATTGATGCGGAAAAGGTACGTAAGAAATTTCTGGGCAAACCAATCGAGGTGTGGAAACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGAGAAAATTCGCGAACATTCTGCAGTTGTTGACATCTTCGAATACGATATTCCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGAGGAAGAACTCAAGATCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAAACGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAGCGCTTTCTCAAAGTTATCCGCGAGAAGGATCCGGACATTATCGTTACTTATAACGGCGACTCTTTTGACTTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAAACTGCCTATCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTATCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTCGTCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCCATGAGATTGCAGAGGCGTGGGAATCCGGTGAGGGCCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCTTCCCAATGGAAATCCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTTTCTCCTGCGCAAAGCGTACGAACGCAACGAACTGGCTCCGAACAAGCCATCTGAACGTGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTATGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAACATCGTGTACCTCGATTTTCGCTCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCTCGAGGGCTGCAAAGAGTATGATGTTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACATCCCGGGCTTTATTCCGTCTCTCCTGGGCCATCTGCTCGAGGAACGCCAAAAGATTAAGCGTAAAATGAAGGCGTCCAAGGATCCGATTGAAAAAATACTGCTCGACTATCGCCAAAGAGCGATTAAACTCCTCGCAAACTCTTTTTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGCTCGTGCGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTAAGTCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGGATTACATTAACTCGAAGCTCCCGGGTCTCCTGGAGCTCGAATATGAAGGCTTTTATAAACGCGGCTTCTTCGTTACCAAGAAGAGATATGCGCTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAAAGTTCTCGAGACTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAGAATTGTAAAAGAAGTAACCCAAAAGCTCGCTAAATATGAAATTCCACCAGAGAAGCTCGCGATTTATGAGCAGATTACTCCCCCCCTGCATGAGTATAAGGCGATTGGTCCCCACGTGGCTGTTGCAAAGAGACTGGCTGCTAGAGGCGTGAAAATTAAACCGGGTATGGTAATAGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATACGATCTGAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGCTTTTGGCTACCGTAAGGAAGACCTCCGCTACCAAAAGACTAAACAGGTTGACCTCACTGCTTGCCTCAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCTCCGAAGGAGCTCCTGCAGATGCTGGAGAAGCAGAAAAAGTGA SEQ ID NO: 6 PhS2 amino acidsequence with the linker and the Sso7d coding region in lower case, andthe linker region in bold.MILDVDYITEEGKPVIRLFKKENGEFKVEYDRTFRPYIYALLKDDSKIDEVRKITGERHGKIVRIIDAEKVRKKFLGKPIEVWKLYFEHPQDVPTIREKIREHSAVVDIFEYDTPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKKIDLPYVEVVSSEREMIKRFLKVIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLPIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWESGEGLERVAKYSMEDAKATYELGKEFFPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPSEREYERRLRESYTGGYVKEPEKGLWENIVYLDFRSLYPSIIITHNVSPDTLNLEGCKIEYDVAPEVGHRFCKDIPGFIPSLLGHLLEERQKIKRKMKASRDPIEKILLDYRQRAIKLLANSFYGYYGYAKARWYCKIECAESVTAWGREYIELVRKELEEKFGFKVLYIDTDGLYATIPGGKSEEIKKKALEFVDYINSKLPGLLELEYEGFYKRGFFVTKKRYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLETILKHGNVEEAVRIVKEVTQKLAKYEIPPEKLAIYEQITPPLHEYKAIGPHVAVAKRLAARGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDLKKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRYQKTKQVDLTACLNIKKSgtggggatvkfkykgeekevdiskikkvwrvgkmisftydegggktgrgavsekdapkellqmlekqkk* SEQ ID NO: 7 PhS5nucleic acid sequence ATGATCCTGGATGCTGACTACATCACTGAAGACGGCAAACCGATTATCCGTCTGTTCAAAAAAGAGAACGGCGAATTTAAGGTTGAGTATGATCGCAACTTTCGTCCATACATTTACGCTCTGCTGAGAGATGATTCTCAGATTGATGAAGTTAAAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCATTGATGCGGAAAAGGTAGAAAAGAAATTTCTGGGCAGACCAATCACCGTGTGGAGACTGTATTTCGAACATCCACAAGATGTTCCGGCTATTCGCGATAAAGTTCGCGAACATCCTGCAGTTGTTGACATCTTCGAATACGATATTCCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGAGGAAGAACTCAAGCTCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAAACGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAACGTTTTCTCAGAGTTATCCGCGAGAAGGATCCGGACATTATCATTACTTATAACGGCGACTCTTTTGACTTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGCCTCTCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTATCGGCGATATGACCGCTGTAGAAATTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTACTCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAGAGGCGTGGGAATCCGGTAAGAACCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCCTCCCAATGGAAATCCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTATCTCCTGCGCAAAGCGTACGAACGCAACGAAGTGGCTCCGAACAAGCCAGACGAAGAAGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTATGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAACCTCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAAAGAGTATGATATTGCTCCGCAAGTAGGCCACAAGTTCTGCAAGGACTTCCCGGGCTTTATTCCGTCTCTCCTGAAACATCTGCTCGATGAACGCCAAGAGATTAAGCGTAAAATGAAGGCGTCCAAGGATCCGATTGAAAAAAAAATGCTCGACTATCGCCAAAGAGCGATTAAACTCCTCGCAAACTCTTTTTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGCTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTAAGCCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGAAATACATTAACTCGAAGCTCCCGGGTCTCCTGGAGCTCGAATATGAAGGCTTTTATGTTCGCGGCTTCTTCGTTACCAAGAAGAGATATGCGGTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAGAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAAAATTGTAAAAGAAGTAACCCAAAAGCTCGCTAAATATGAAATTCCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAGACTGGCTGCTAGAGGCGTGAAAGTTAGACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATACGATCTGAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGCTTTTGGCTACCGTAAGGAAGACCTCCGCTGGCAAAAGACTAAACAGGTTGGCCTCACTTCTTGGCTCAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGTGA SEQ ID NO: 8 PhS5 polypeptidesequence with the linker and the Sso7d coding region in lower case, andthe linker region in bold.MILDADYITEDGKPIIRLFKKENGEFKVEYDRNFRPYIYALLRDDSQIDEVKKITAERHGKIVRIILDAEKVEKKFLGRPITVWRLYFEHPQDVPAIRDKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKLLAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKKIDLPYVEVVSSEREMIKRFLRVIREKDPDIIITYNGDSFDFPYLAKRAEKLGIKLPLGRDGSEPKMQRIGDMTAVEIKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAEAWESGNLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNEVAPNKPDEEEYERRLRESYTGGYVKEPEKGLWENLVSLDFRALYPSIIITHNVSPDTLNREGCKEYDIAPQVGHKFCKDFPGFIPSLLKHLLDERQEIKRKMKASRDPIEKKMLDYRQRAIKLLANSFYGYYGYAKARWYCKECAESVTAWGREYIELVWKELEEKFGFKVLYIDTDGLYATIPGGKPEEIKKKALEFVKYINSKLPGLLELEYEGFYVRGFFVTKKRYAVIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVKIVKEVTQKLAKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISNRAILAEEYDLKKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQVGLTSWLNIKKSgtggggatvkfkykgeekevdiskikkvwrvgkmisftydegggktgrgavsekdapkellqmlekqkk* SEQ ID NO: 9 PhS7 nucleicacid sequence ATGATCCTGGATGCTGACTACATCACTGAAGACGGCAAACCGATTATCCGTCTGTTCAAAAAAGAGAACGGCGAATTTAAGGTTGAGTATGATCGCAACTTTCGTCCATACATTTACGCTCTGCTGAGAGATGATTCTCAGATTGATGAAGTTAAAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCATTGATGCGGAAAAGGTAGAAAAGAAATTTCTGGGCAGACCAATCACCGTGTGGAGACTGTATTTCGAACATCCACAAGATGTTCCGGCTATTCGCGATAAAGTTCGCGAACATCCTGCAGTTGTTGACATCTTCGAATACGATATTCCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCATGGAGGGCGAGGAAGAACTCAAGCTCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAAACGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAACGTTTTCTCAGAGTTATCCGCGAGAAGGATCCGGACATTATCATTACTTATAACGGCGACTCTTTTGACTTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGCCTCTCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTATCGGCGATATGACCGCTGTAGAAATTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTACTCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAGAGGCGTGGGAATCCGGTAAGAACCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCCTCCCAATGGAAATCCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTATCTCCTGCGCAAAGCGTACGAACGCAACGAAGTGGCTCCGAACAAGCCAGACGAAGAAGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTATGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAACCTCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAGAAACTATGATGTTGCTCCGCAAGTAGGCCACAAGTTCTGCAAGGACTTCCCGGGCTTTATTCCGTCTCTCCTGGGCCGTCTGCTCGAGGAACGCCAAGAGATTAAGACTAAAATGAAGGCGACCAAGGATCCGATTGAAAAAAAACTGCTCGACTATCGCCAAAAAGCGATTAAAATCCTCGCAAACTCTTTTTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCAAATACATCGAGTTCGTGCGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTAAGCCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGAAATACATTAACTCGAAGCTCCCGGGTCTCCTGGAGCTCGAATATGAAGGCTTTTATGTTCGCGGCTTCTTCGTTACCAAGAAGAGATATGCGGTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAGAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAAAATTGTAAAAGAAGTAACCCAAAAGCTCGCTAAATATGAAATTCCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAGACTGGCTGCTAGAGGCGTGAAAGTTAGACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATACGATCTGAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGCTTTTGGCTACCGTAAGGAAGACCTCCGCTGGCAAAAGACTAAACAGGTTGGCCTCACTTCTTGGCTCAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGTGA SEQ ID NO: 10 PhS7 polypeptidesequence with the linker and the Sso7d coding region in lower case, andthe linker region in bold.MILDADYITEDGKPIIRLFKIKENGEFKVEYDRNFRPYIYALLRDDSQIDEVKKITAERHGKIIVRIIDAEKVERKFLGRPITVWRLYFEHPQDVPAIRDKVREHPAVVDIFEYDTPFAKRYLIDKGLIIPMEGEEELKLLAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKKIDLPYVEVVSSERFMIKRFLRVIREKDPDIIITYNGDSFDFPYLAKRAEKLGIKLPLGRDGSEPKMQRIGDMTAVEIKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAEAWESGKNLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNEVAPNKPDEEEYERRLRESYTGGYVKEPEKGLWENLVSLDFRALYPSIIITHNVSPDTLNREGCRNYDVAPQVGHKFCKDFPGFIPSLLGRLLEERQEIKTKMKATKDPIEKKLLDYRQKAIKILANSFYGYYGYAKARWYCKECAESVTAWGRKYIEFVRKELEEKFGFKVLYIDTDGLYATIPGGKPEEIKKKALEFVKYINSKLPGLLELEYEGFYVRGFFVTKKRYAVIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVKIVKEVTQKLAKYEIIPPEKLAIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISNRAILAEEYDLKKHKYDAIEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQVGLTSWLNIKKSgtggggatvkfkykgeekevdiskikkvwrvgkmisftydegggktgrgavsekdapkellqmlekqkk* SEQ ID NO: 11 Hyb1nucleic acid sequence ATGATCCTGGATGCTGACTACATCACTGAAGACGGCAAACCGGTTATCCGTCTCTTCAAAAAAGAGAACGGCGAATTTAAGATTGAGTATGATCGCACCTTTCGTCCATACATTTACGCTCTGCTGAGAGATGATTCTAAGATTGAGGAAGTTAGAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGTGGAAAAGGTAAGGAAGAAATTTCTGGGCAGACCAATCAAGGTGTGGAGACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGATAAAGTTCGCGAACATCCTGCAGTTATTGACATCTTCGAATACGATATTGCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGAGGAAGAACTCAAGATCCTGGCGTTCGATATAGAAACCCTCTATCACGGAAGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAAACGAAGCAAAGGTGATTACTTGGAAAAACATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAACGCTTTCTCAGAATTATCCGCGAGAAGGATCCGGACATTATCGTTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGACTCTCGGCCGTGATGGTTGCGAGGCGAAGATGCAGCGTCTCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATTATGTAATTAGCCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGATATTGCAGAGGCGTGGGAAACCGGTAAGGGCCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCCTCCCAATGGAAGCTCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTATCTCCTGCGCAAAGCGTACGAACGCAACGAAGTGGCTCCGAACAAGCCATACGAACGAGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAGCCTCGTGTCCCTCGATTTTCGCTCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAAAGACTATGATATTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCTTGGCTTTATTCCGTCTCTCCTGGGGCATCTGCTCGAGGAACGCCAAGAGATTAAGACCAAAATGAAGGAGACCCANGATCCGATTGAAAAAATACTGCTCGACTATCGCCAAAAAGCGATTAAACTCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTGAGCCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGAAATACATTAACTCGAAGCTCCCCGGTCTCTTGGAGCTCGAATATGAAGGCTTTTATAAGCGCGGCTTCTTCGTTACCAAGAAGAGATATGCGGTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAAAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAAAATTGTAAAAGAAATAATCGAAAAGCTCGCTAAATATGAAATACCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAAACTGGCTGCTAGAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAAACGTGCAATTCTAGCTGAGGAATTCGATCCGAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGTTGGCAAAAGACTAAACAGGCTGGCCTCACTGCTTGGCTCAACATTAAAAAATCCGGTACCCACTAG SEQ ID NO: 12 Hyb1 amino acidsequence MILDADYITEDGKPVIRLFKKENGEFKIEYDRTFRPYIYALLRDDSKIEEVRKITAERHGKIVRWDVEKVRKKFLGRPIKVWRLYFEHPQDVPTIRDKVREHPAVIDIFEYDIAFAKRYLIDKGLIPMEGEEELKILAFDIETLYHGSEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIIKRFLRIIREKDPDIIVTYNGDSFDLPYLAKRAEKLGIKLTLGRDGCEAKMQRLGDMTAVEVKGRIHFDLYYVISRTTNLPTYTLEAVYEAIFGKPKEKVYADDIAEAWETGKGLERVAKYSMEDAKATYELGKEFLPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNEVAPNKPYEREYERRLRESYTGGFVKEPEKGLWESLVSLDFRSLYPSIIITHNVSPDTLNREGCKDYDIAPEVGHKFCKDFLGFIPSLLGHLLEERQEIKTKMKFTXDPIELKILLDYRQKAIKLLANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVWKELEEKFGFKVLYIDTDGLYATIPGGEPEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEIIEKLAKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAARGVKIKPGMVIGYIVLRGDGPISKRAILAEEFDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRWQKTKQAGLTAWLNIKKS* SEQ ID NO: 13 HyS1 (Hyb1 with Sso7d at theC-terminus) nucleic acid sequenceATGATCCTGGATGCTGACTACATCACTGAAGACGGCAAACCGGTTATCCGTCTCTTCAAAAAAGAGAACGGCGAATTTAAGATTGAGTATGATCGCACCTTTCGTCCATACATTTACGCTCTGCTGAGAGATGATTCTAAGATTGAGGAAGTTAGAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGTGGAAAAGGTAAGGAAGAAATTTCTGGGCAGACCAATCAAGGTGTGGAGACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGATAAAGTTCGCGAACATCCTGCAGTTATTGACATCTTCGAATACGATATTGCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGAGGAAGAACTCAAGATCCTGGCGTTCGATATAGAAACCCTCTATCACGGAAGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAAACGAAGCAAAGGTGATTACTTGGAAAAACATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAACGCTTTCTCAGAATTATCCGCGAGAAGGATCCGGACATTATCGTTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGACTCTCGGCCGTGATGGTTGCGAGGCGAAGATGCAGCGTCTCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATTATGTAATTAGCCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGATATTGCAGAGGCGTGGGAAACCGGTAAGGGCCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCCTCCCAATGGAAGCTCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTATCTCCTGCGCAAAGCGTACGAACGCAACGAAGTGGCTCCGAACAAGCCATACGAACGAGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAGCCTCGTGTCCCTCGATTTTCGCTCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAAAGACTATGATATTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCTTGGCTTTATTCCGTCTCTCCTGGGGCATCTGCTCGAGGAACGCCAAGAGATTAAGACCAAAATGAAGGAGACCCANGATCCGATTGAAAAAATACTGCTCGACTATCGCCAAAAAGCGATTAAACTCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTGAGCCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGAAATACATTAACTCGAAGCTCCCCGGTCTCTTGGAGCTCGAATATGAAGGCTTTTATAAGCGCGGCTTCTTCGTTACCAAGAAGAGATATGCGGTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAAAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAAAATTGTAAAAGAAATAATCGAAAAGCTCGCTAAATATGAAATACCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAAACTGGCTGCTAGAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAAACGTGCAATTCTAGCTGAGGAATTCGATCCGAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGTTGGCAAAAGACTAAACAGGCTGGCCTCACTGCTTGGCTCAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGTGA SEQ ID NO: 14 HyS1 (Hyb1 with Sso7dat the C-terminus) polypeptide sequence with the linker and the Sso7dcoding region in lower case, and the linker region in bold.MILDADYITEDGKPVIRLFKKENGEFKIEYDRTFRPYIYALLRDDSKIEEVRKITAERHGKIVRIVDVEKVRKRFLGRPIKVWRLYFEHPQDVPTIRDKVREHPAVIDIFEYDIAFAKRYLIDKGLIPMEGEEELKILAFDIETLYHGSEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDLPYLAKRAEKLGIKLTLGRDGCEAKMQRLGDMTAVEVKGRIHFDLYYVISRTINLPTYTLEAVYEAIFGKPKVYADDIAEAWETGKGLERVAKYSMEDAKATYELGKEFLPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNFVAPNKPYEREYERRLRESYTGGFVKEPEKGLWESLVSLDFRSLYPSIIITHNVSPDTLNREGCKDYDIAPEVGHKFCKDFLGFIPSLLGHLLEERQEIKTKMKETXDPIEKILLDYRQKAIKLLANSYYGYYGYARARWYCKECAESVTAWGREYIEFVWKELEEKFGFKVLYIDTDGLYATIPGGEPEEIIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVMIVKEIIEKLAKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAARGVKIKPGMVIGYIVLRGDGPISKRAILAEEFDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRWQKTKQAGLTAWLNIKKSgtggggatvkflykgeekevdiskikkvwrvgkmisftydegggktglgavsekdapkellqmlekqkk* SEQ ID NO: 15 Hyb2(premature stop codon in bold) nucleic acid sequenceATGATCCTGGATGCTGACTACATCACTGAAGAAGGCAAACCGGTTATCCGTATCTTCAAAAAAGAGAACGGCGAATTTAAGGTTGAGTATGATCGCAACTTTCGTCCATACATTTACGCTCTGCTGGAAGATGATTCTAAGATTGATGAAGTTAGAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGCGGAAAAGGTAGAGAAGAAATTTCTGGGCAGACCAATCACGGTGTGGAAACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGAGAAAATTCGCGAACATTCTGCAGTTGTTGGCATCTTCGAATACGATATTCCATTTGCAAAGAGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGAGGAAGAACTCAAGCTCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGCTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAGACGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAGCGCTTTCTCAGAGTTATCCGCGAGAAGGATCCGGACGTTATCGTTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAXACTCGGTATTAAACTGCCTCTCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTCTCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTAGCCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAGGGGCGTGGGAAACCGGTGAGGACCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGATTTATGAACTCGGCAAAGAATTCTTCCCAATGGAAGTTCAGCTCCCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTTGCTCCTGCGCAAAGCGTACGAACGCAACGAACTGGCTCCGAACAAGCCAGCCGAACAAGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAGACCTCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAAAGACTATGATATTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCTTGGCTTTATTCCGTCTCTCCTGGGGCATCTGCTCGAGGAACGCCAAGAGATTAAGACCAAAATGAAGGAGACCCANGATCCGATTGAAAAAATACTGCTCGACTATCGCCAAAAAGCGATTAAACTCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTGAGCCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGAAATACATTAACTCGAAGCTCCCCGGTCTCTTGGAGCTCGAATATGAAGGCTTTTATAAGCGCGGCTTCTTCGTTACCAAGAAGAGATATGCGGTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAAAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAAAATTGTAAAAGAAATAATCGAAAAGCTCGCTAAATATGAAATACCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAAACTGGCTGCTAGAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATTCGATCTGAGAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGTTAGCAAAAGACTAAACAGGCTGGACTCACTGCTTGGCTCATCATTAAAAAATCCGGTACCCACTAGTGC SEQ ID NO: 16 Hyb2 polypeptidesequence MILDADYITEEGKPVIRIFKKENGEFKVEYDRNFRPYIYALLEDDSKIDEVRKITAERHGKIVRIVDAEKVEKKFLGRPITVWKLYFEHPQDVPTIREKIREHSAVVGIFEYDIPFAKSYLIDKGLIPMEGEEELKLLAFDIETLYHEGEEFAKGPIIMISYADEDEAKVITWKKIDLPYVEVVSSEREMIKRFLRVIREKDPDVIVTYNGDSFDLPYLAKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEVKGRIHFDLYHVISRTINLPTYTLEAVYEAIFGKPKEKVYADEIAGAWETGEDLERVAKYSMEDAKAIYELGKEFFPMEVQLPRLVGQPLWDVSRSSTGNLVEWLLLRKAYERNFLAPNKPNEQEYERRLRESYTGGFVREPEKGLWEDLVSLDFRALYPSIIITHNVSPDTLNREGCKDYDIAPEVGHKFCKDFLGFIPSLLGHLLEERQEIKTKMKETXDPIEKILLDYRQKAIKLLANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVWKELEEKFGFKVLYIDTDGLYATIPGGEPEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKIITRGLEIVRRDWSEIAKIETQAKVLEAILKHGNVEEAVKIVKEIIEKLAKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAARGVKIKPGMVIGYIVLRGDGPISNRAILAEEFDLRKHKYDAEYYIENQVLPAVLRILEGF GYRKEDLR* SEQ ID NO:17 Hyb3 (premature stop codon in bold) nucleic acid sequenceATGATCCTGGATGCTGACTACATCACTGAAGAAGGCAAACCGGTTATCCGTATCTTCAAAAAAGAGAACGGCGAATTTAAGGTTGAGTATGATCGCAACTTTCGTCCATACATTTACGCTCTGCTGGAAGATGATTCTAAGATTGATGAAGTTAGAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGCGGAAAAGGTAGAGAAGAAATTTCTGGGCAGACCAATCACGGTGTGGAAACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGAGAAAATTCGCGAACATTCTGCAGTTGTTGGCATCTTCGAATACGATATTCCATTTGCAAAGAGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGAGGAAGAACTCAAGCTCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGCTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAGACGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAGCGCTTTCTCAGAGTTATCCGCGAGAAGGATCCGGACGTTATCGTTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGCCTCTCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTCTCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTAGCCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAGGGGCGTGGGAAACCGGTGAGGACCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGATTTATGAACTCGGCAAAGAATTCTTCCCAATGGAAGTTCAGCTCCCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTTGCTCCTGCGCAAAGCGTACGAACGCAACGAACTGGCTCCGAACAAGCCAGCCGAACAAGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAGACCTCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAAAGACTATGATATTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCTTGGCTTTATTCCGTCTCTCCTGGGGCATCTGCTCGAGGAACGCCAAGAGATTAAGACCAAAATGAAGGAGACCCANGATCCGATTGAAAAAATACTGCTCGACTATCGCCAAAAAGCGATTAAACTCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTGAGCCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGAAATACATTAACTCGAAGCTCCCCGGTCTCTTGGAGCTCGAATATGAAGGCTTTTATAAGCGCGGCTTCTTCGTTACCAAGAAGAGATATGCGGTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAAAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAAAATTGTAAAGAAATAATCGAAAAGCTCGCTAAATATGAAAATACCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAAACTGGCTGCTAGAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATTCGATCTGAGAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGTTAGCAAAAGACTAAACAGGCTGGACTCACTGCTTGGCTCATCATTAAAAAATCCGGTACCCACTAGTGC SEQ ID NO: 18 Hyb3 polypeptidesequence MILDADYITEEGKPVIRIFKKENGEFKVEYDRNFRPYIYALLEDDSKIDEVRKITAERHGKIVRIVDAEKVEKKFLGRPITVWKLYFEHPQDVPTIREKIREHSAVVGIFEYDIPFAKSYLIDKGLIPMEGEEELKLLAFDIETLYHEGEEFAKGPIIMISYADEDEAKVITWKKIDLPYVEVVSSEREMIKRFLRVIREKDPDVIVTYNGDSFDLPYLAKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEVKGRIHFDLYHVISRTINLPTYTLEAVYEAIFGKPKEKVYADEIAGAWETGEDLERVAKYSMEDAKAIYELGKEFFPMEVQLPRLVGQPLWDVSRSSTGNLVEWLLLRKAYERNELAPNKPAEQEYERRLRESYTGGFVKEPEKGLWEDLVSLDFRALYPSIIITHNVSPDTLNREGCKDYDIAPEVGHKFCKDFLGFIPSLLGHLLEERQEIKTKMKETXDPIEKILLDYRQKAIKLLANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVWKRLEEKFGFKVLYIDTDGLYATIPGGEPEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEIIEKLAKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAARGVKIKPGMVIGYIVLRGDGPISNRAILAEEFDLRKHKYDAEYYIENQVLPAVLRILEGFG YRKEDLR* SEQ ID NO:19 HyS4 (with Sso7d at the C-terminus) nucleic acid sequenceATGATCCTGGATGCTGACTACATCACTGAAGAAGGCAAACCGGTTATCCGTATCTTCAAAAAAGAGAACGGCGAATTTAAGGTTGAGTATGATCGCAACTTTCGTCCATACATTTACGCTCTGCTGGAAGATGATTCTAAGATTGATGAAGTTAGAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGCGGAAAAGGTAGAGAAGAAATTTCTGGGCAGACCAATCACGGTGTGGAAACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGAGAAAATTCGCGAACATTCTGCAGTTGTTGGCATCTTCGAATACGATATTCCATTTGCAAAGAGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGAGGAAGAACTCAAGCTCCTGGCGTTCGATATAGAAACCCTCTATCACGAAGGCGAAGAGTTTGCTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAGACGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAGCGCTTTCTCAGAGTTATCCGCGAGAAGGATCCGGACGTTATCGTTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGCCTCTCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTCTCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTAGCCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAGGGGCGTGGGAAACCGGTGAGGACCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGATTTATGAACTCGGCAAAGAATTCTTCCCAATGGAAGTTCAGCTCCCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTTGCTCCTGCGCAAAGCGTACGAACGCAACGAACTGGCTCCGAACAAGCCAGCCGAACAAGAGTATGAACGCCGTCTCCGCGAGTCTTACACTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAGACCTCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAAAGACTATGATATTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCTTGGCTTTATTCCGTCTCTCCTGGGGCATCTGCTCGAGGAACGCCAAGAGATTAAGACCAAAATGAAGGAGACCCANGATCCGATTGAAAAAATACTGCTCGACTATCGCCAAAAAGCGATTAAACTCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTGAGCCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGAAATACATTAACTCGAAGCTCCCCGGTCTCTTGGAGCTCGAATATGAAGGCTTTTATAAGCGCGGCTTCTTCGTTACCAAGAAGAGATATGCGGTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAAAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAAAATTGTAAAAGAAATAATCGAAAAGCTCGCTAAATATGAAATACCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAAACTGGCTGCTAGAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAAACGTGCAATTCTAGCTGAGGAATTCGATCCGAAAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGTTGGCAAAAGACTAAACAGGCTGGCCTCACTGCTTGGCTCAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGTGA SEQ ID NO: 20 HyS4 (with Sso7d atthe C-terminus) polypeptide sequence with the linker and the Sso7dcoding region in lower case, and the linker region in bold.MILDADYITEEGKPVIRIFKENGEFKVEYDRNFRPYIYALLEDDSKIDEVRKITAERHGKIVRIVDAEKVEKKFLGRPITVWKLYFEHPQDVPTIREKIREHSAVVGIFEYDIPFAKSYLIDKGLIPMEGEEELKLLAFDIETLYHEGEEFAKGPITMISYADEDEAKVITWKKIDLPYVEVVSSEREMTKRFLRVIREKDPDVIVTYNGDSFDLPYLAKPAEKLGIKLPLGRDGSEPKMQRLGDMTAVEVKGRIHFDLYHVISRTTNLPTYTLEAVYEAIFGKPKEKVYADEIAGAWETGEDLERVAKYSMEDAKAIYELGKIEFFPMEVQLPRLVGQPLWDVSRSSTGNLVEWLLLRKAYERNIELAPNKPAEQEYERRLRESYTGGFVKEPEKGLWEDLVSLDFRALYPSIIITHNVSPDTLNRFGCRDYDIAPEVGHRFCKDFLGFIPSLLGHLLEERQEIKTRMKFTXDPIEKILLDYRQKAIKLLANSYYGYYGYAKARWYCKLCAESVTAWGREYIEFVWKELEEKFGFKVLYIDTDGLYATIPGGEPEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEIIEKLAKYEILPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAARGVKIKPGMVIGYIVLRGDGPISKRAILAEEFDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRWQKTKQAGLTAWLNIKKSgtggggatvkfkykgeekevdiskikkvwrvgkmisftydegggktgrgavsekdapkellqmlekqkk* SEQ ID NO: 21: Sso7dcoding region: ACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAGTGA SEQ ID NO: 22 Sso7d bindingdomain: ATVKFKYKGEEKLVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDA PKELLQMLEKQKKSEQ ID NO: 23 signature amino acid sequence common to polymerases of theinvention YGYYGYAKARWYCRECAESVTAWGR SEQ ID NO: 24 Parent Pyrococcusfuriosus (Pfu) polymerase polypeptide sequenceMILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTTNLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS SEQ ID NO: 25 parent Pyrococcus sp. GD-B (DeepVent®) polymerase polypeptide sequenceMILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVRKITAERHGKIVRIIDAEKVRKKFLGRPIEVWRLYFEHPQDVPAIRDKIREHSAVIDIFEYDPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEEAKVITWKKIDLPYVEVVSSEREMIKRFLKVIITEKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAGGYVKEPEKGLWEGLVSLDFRSLYPSIIITHNVSPDTLNREGCREYDVAPEVGHKCRDFPGFIPSLLKRLLDERQEIKRKMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEERFGFKVLYIDTDGLYATIPGAKPEEIKKKALEFVDYINAKLPGLLELEYEGFYVRGFFVTKKYALIDEEGKIITRGLEIVRDWSEIKETQAKVLEAILKKGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDLRKHKYDAEYYIENQVLPAVLRILEMGYRKED LRWQKTKQTGLTAWLNIKKKSEQ ID NO: 26 Amino acid sequences of designed hybrid protein The“X” residue represents a hybrid protein position that is encoded by adegeneracy. The residue at that position is typically either that of thePfu parent or the Deep VenT® parent.MILDXDYITEXGKPXIRXFKKENGXFKXEXDRXFRPYIYALLXDDSXIXEVXKITXERHGKIVRIXDXEKVXRRFLGXPIXVWXLYXEHPQDVPXRXKXREHXAVXDIFEYDTPFAKRYLTDKGLIPMEGXEELKXLAFDIETLYHEGEEFXKGPITMISYADEXEAKVITWKXIDLPYVEVVSSEREMIKRFLXXIREKDPDXIXTYNGDSFDXPYLXRRAEKLGIKLXXGRDGSEPKMQRXGDMTAVEXKGRIIIFDLYHVIXRTINLPTYTLEAVYEATFGKPKEKVYAXEIAXAWEXGXXLERVAKYSMEDAKXTYELGXEFXPMEXQLSRLVGQPLWDVSRSSTGNLVEWXLLRKAYERNEXAPNKPXEXEYXRRLRESYXGGXVKEPEKGLWEXXVXLDFRXLYPSIIITHNVSPDTLNXEGCXXYDXAPXVGHKFCKDXPGFIPSLLXXLLXERQXIIKXKMKXXXDPIEKXXLDYRQXAIKXLANSXYGYYGYAKARWYCKECAESVTAWGRXYIEXVXKELEEKFGFKVLYIDTDGLYATIPGXXXEEIKKKALEFVKYINXKLPGLLELEYEGFYXRGFFVTKKXYAMDEEGKXITRGLEIVRRDWSEIAKETQAXVLEXILKHGXVEEAVXIVKLEVXXKLXXYEIPPEKLXIYEQITRPLHEYKAIGPHVAVAKXLAAXGVKXXPGMVIGYIVLRGDGPISXRAILAEEXDXXKHKYDAEYYIENQVLPAVLRILEXFGYRKEDLRXQKTXQXGLTXWLNIKKS

1. A hybrid polymerase that comprises an amino acid sequence at least94% identical to the amino acid sequence set forth in SEQ ID NO:2,wherein the polymerase has an increased ratio of polymerase activity toexonuclease activity relative to Pyrococcus furiosus polymerase orPyrococcus sp. GD-B polymerase.
 2. The hybrid polymerase of claim 1,wherein the polymerase further comprises a sequence non-specificdouble-stranded nucleic acid DNA binding domain joined to thepolymerase, wherein the sequence non-specific double-stranded nucleicacid binding domain has at least 75% identity to the amino acid sequenceset forth in SEQ ID NO:22.
 3. The hybrid polymerase of claim 1, whereinthe polymerase further comprises a sequence non-specific double-strandednucleic acid DNA binding domain joined to the polymerase, wherein thesequence non-specific double-stranded nucleic acid binding domain isselected from the group consisting of Sso7d, Sac7d, and Sac7e.
 4. Thehybrid polymerase of claim 1, wherein the polymerase has the amino acidsequence set forth in SEQ ID NO:4.
 5. An isolated nucleic acid encodinga hybrid polymerase that comprises an amino acid sequence at least 94%identical to the amino acid sequence set forth in SEQ ID NO:2, whereinthe polymerase has an increased ratio of polymerase activity toexonuclease activity relative to Pyrococcus furiosus polymerase orPyrococcus sp. GD-B polymerase.
 6. The isolated nucleic acid of claim 5,wherein the polymerase encoded by the nucleic acid further comprises asequence non-specific double-stranded nucleic acid DNA binding domainjoined to the polymerase, wherein the sequence non-specificdouble-stranded nucleic acid binding domain has at least 75% identity tothe amino acid sequence set forth in SEQ ID NO:22.
 7. The isolatednucleic acid of claim 5, wherein polymerase encoded by the nucleic acidfurther comprises a sequence non-specific double-stranded nucleic acidDNA binding domain joined to the polymerase, wherein the sequencenon-specific double-stranded nucleic acid binding domain is selectedfrom the group consisting of Sso7d, Sac7d, and Sac7e.
 8. The isolatednucleic acid of claim 5, wherein the polymerase encoded by the nucleicacid has the amino acid sequence set forth in SEQ ID NO:4.
 9. Anexpression vector comprising the nucleic acid of claim
 5. 10. A hostcell comprising the expression vector of claim
 9. 11. A method ofamplifying a target sequence using a hybrid polymerase, the methodcomprising the steps of: providing a polymerase according to claim 1,combining the polymerase in an amplification reaction mixture thatcomprises components for the amplification of the target sequence, andamplifying the target sequence.
 12. A hybrid polymerase havingpolymerase activity, wherein the polymerase comprises SEQ ID NO:23 andis at least 90% identical over 700 contiguous amino acids of thePyrococcus furiosus (Pfu) polymerase sequence set forth in SEQ ID NO: 24or at least 90% identical over 700 contiguous amino acids of thePyrococcus sp. GB-D polymerase sequence set forth in SEQ ID NO:25, withthe proviso that (a) when the polymerase is at least 90% identical toSEQ ID NO:24, the sequence comprises at least twenty hybrid positionsthat are mutated from the native Pfu residue to the residue that occursat the corresponding position of SEQ ID NO:25, wherein the hybridposition is one of the residues designated as “X” in SEQ ID NO:26; or(b) when the polymerase is at least 90% identical to SEQ ID NO:25, thesequence comprises at least twenty hybrid mutations that are mutatedfrom the native Pyrococcus sp. GB-D residue to the residue that occursat the corresponding position of SEQ ID NO:24, wherein the hybridposition is one of the residues designated as “X” in SEQ ID NO:26; andfurther, wherein the hybrid polymerase has an increased ratio ofpolymerase activity to exonuclease activity relative to Pyrococcusfuriosus or Pyrococcus sp. GB-D.